METHODS

Subjects

All patients were ascertained for study based upon the presence of SHFM in one or more limbs. Informed consent was obtained before participation, and these studies were approved by the Institutional Review Board of Self Memorial Hospital (Greenwood, SC). All patients were examined by a clinical geneticist. A diagnosis of EEC syndrome was made if at least two of the following features were present: (1) SHFM, (2) ectodermal dysplasia affecting the hair, nails, teeth, lacrimal ducts, sweat glands, or mammary glands, and (3) clefting of the lip and/or palate. Classification of cases as syndromic or non-syndromic SHFM was based on the presence or absence of extraskeletal abnormalities. Patients with isolated limb defects consistent with SHFM were classified as having non-syndromic SHFM type I according to the classification of Zlotogora.⁴ Patients with SHFM associated with hypoplasia or absence of one or more long bones (tibia, fibula, femur, radius, ulna, humerus) were classified as having non-syndromic SHFM type II, also known as SHFM with long bone deficiency (SHFLD).¹⁴

Clinical phenotypes

EEC patients

All 10 patients had at least two major findings of EEC syndrome. One case, ALT, was familial with an affected mother and two affected sons. Two patients also had colobomatous microphthalmia and microcephaly, one had hydrocephalus, and another had seizures, mental retardation, and blindness (table 1).

SHFM patients

Among the 24 SHFM patients, six sporadic cases had SHFM while 16 patients, seven familial and nine sporadic, had SHFM associated with long bone deficiency (SHFLD) (SHFM type II). An additional two sporadic cases had syndromic SHFM, including one with nystagmus (Karsch-Neugebauer syndrome (MIM183800)) and one with seizures and mental retardation (table 2).

DNA extraction and SSCP analysis

DNA from peripheral blood leucocytes was extracted using a high salt precipitation procedure.³¹ Mutation analysis was performed using intronic primers that were suitable for amplification of exons 1 to 15 of P63.^11,27 PCR was performed in a total volume of 10 μl, using 50 ng genomic DNA. The reactions were carried out at a final concentration of 50 mmol/l KCl, 1.5 mmol/l MgCl₂, 1 μmol/l of each primer, 50 μmol/l dNTPs, 1 μCi α³²P dCTP, and 0.5 units of Taq DNA polymerase (Sigma, St Louis, MO, USA). PCR conditions were as follows: one cycle at 95°C for four minutes; 20 cycles at 95°C for 30 seconds, annealing at 65°C with a decrement of 0.5°C/cycle for 30 seconds, and elongation at 72°C for 30 seconds; 15 cycles at 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; and one cycle at 72°C for five minutes. Amplification was carried out in an MJ Research thermocycler (MJ PTC-2000); 5 μl of the ³²P labelled PCR product was denatured in loading buffer (95% formamide, 10 mmol/l NaOH, 0.25% bromophenol blue, 0.25% xylene cyanol), and analysed on a 0.5 × MDE (FMC, Rockland, ME, USA) gel at 8 W for 18 hours. The radioactive signal was visualised using Biomax MS film (Kodak, Rochester, NY) after two hours of exposure at −80°C.

Sequencing

Both strands of the PCR products generated from the patients and a normal control DNA were sequenced using the Thermosequenase Cy™5.5 Dye Terminator kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with exon specific primers covering 30 to 50 bp of flanking intronic sequences.^11,27 Electrophoresis and analysis were performed on an Automated Laser Fluorescence (ALF) DNA sequencer after purification with Autoseq columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Mutation confirmation by restriction digest

The point mutations G728T and G728A removed a BanI site in exon 5. The 291 bp PCR product corresponding to exon 5 was digested with BanI, producing two fragments for controls (194 bp and 97 bp) and three fragments for each patient (291 bp, 194 bp, and 97 bp). The point mutation G953A removed an AciI site in exon 7. The PCR product of 253 bp corresponding to exon 7 was digested with AciI, producing two fragments for controls (143 bp and 110 bp) and three fragments for the patient (253 bp, 143 bp, and 110 bp).

The point mutations G1028C, G1028A, and C1027T removed an MspI site in exon 8. The 260 bp PCR product corresponding to exon 8 was digested with MspI, producing two fragments for controls (157 bp and 103 bp) and three fragments for each patient (260 bp, 157 bp, and 103 bp). All fragments were resolved by electrophoresis on a 2% Seakem™ LE agarose gel (BMA, Rockland, ME, USA).

Polymorphism studies

Base changes were classified as polymorphisms if they fulfilled at least one of the following criteria: (1) the change was also present in the normal white subject who was used as our sequencing control, or (2) the change was not present in an affected parent.

RESULTS

P63 mutation analysis in EEC patients

Screening of the DNA binding domain (exons 5 to 10) of P63 showed seven missense mutations in seven out of 10 unrelated patients with EEC syndrome, including two previously unreported mutations found in patients RG and SH. Patient RG has symmetrical split hand malformations with syndactyly of the thumb (digit 1) and the index finger (digit 2) with separate nails, absent digit 3 with a shallow cleft of the hand, and separate digits 4 and 5. Her left foot has a short second toe with a hyperconvex nail and partial syndactyly of the first and second toes. She also has inverted nipples, thin hair, lacrimal duct stenosis, and a short nasal septum but no clefting of the lip and/or palate (fig 1A).

Patient RG had an abnormal SSCP pattern in exon 5 (fig 2A). Sequence analysis showed a G to T transversion at position 728 (G728T), which predicts an amino acid substitution, arginine for leucine, at position 204 (R204L) (fig 2B). The G728T alteration removes a BanI site and the restriction digest confirmed the mutation (fig 2C). This patient is a sporadic case and genetic analysis of both parents showed the mutation to be de novo (fig 2C). The mutation was not detected in 192 control chromosomes.

Patient SH has bilateral split hand malformations with absence of digits 2 and 3. Her right foot has a wide cleft with an absent second toe and syndactyly of the remaining toes and her left foot has syndactyly of toes 1/2 and 4/5. She also has cleft palate, anodontia, and macrostomia. She is severely mentally retarded (IQ=8) and has a seizure disorder. She is blind in the left eye and has corneal opacity of the right eye (fig 1B). She had meningitis at the age of 10 months.

Patient SH had an altered SSCP pattern of exon 8 (fig 3A). Sequence analysis showed a G to C transversion at position 1028 (G1028C), which predicts an amino acid substitution, arginine to proline, at position 304 (R304P) (fig 3B). This mutation results in the loss of an MspI site in the patient, which made it possible to confirm the mutation by restriction digest (fig 3C). This patient is a sporadic case whose parents were not available for genetic testing. The mutation was not detected in 192 control chromosomes.

Five other patients with typical EEC syndrome also had P63 mutations. All have been reported previously as disease causing mutations in EEC syndrome.^11,27 These include a G to A transition at nucleotide 728 (G728A) which predicts an arginine to glutamine substitution at position 204 (R204Q); a G to A transition at position 953 (G953A) which predicts an arginine to histidine change at position 279 (R279H); a G to A transition at position 1028 (G1028A) which predicts an arginine to glutamine change at position 304 (R304Q) (two patients); and a C to T transition at position 1027 (C1027T) which predicts an arginine to tryptophan change at position 304 (R304W) (table 1).

In the three EEC patients lacking mutations in the DNA binding domain of P63, the analysis was extended to the entire coding region and flanking intronic sequences and no mutations were detected. Interestingly, all three patients had central nervous system abnormalities. Two of these patients had colobomatous microphthalmia and microcephaly, and the third patient had hydrocephalus.

P63 mutation analysis in SHFM patients

SSCP analysis of the entire coding region and flanking intronic sequences of P63 did not detect any mutations in our panel of 24 patients with various forms of SHFM (table 2). However, six different polymorphisms were discovered. In one patient with SHFM type I, a 2 bp deletion was found in intron 2, 8 bp from the splice acceptor site (IVS29-8delAT). The affected mother did not carry this change and RT-PCR analysis using primers located in exons 1 and 4 respectively did not show any aberrant splice forms in the patient carrying the deletion (data not shown). Two SHFM type II patients and one SHFM type I patient had a T to G change 35 bp after the intron 5 splice acceptor site (IVS5+35T>G). This change was seen in a control and an unaffected family member of one patient. A sporadic SHFM type I and a type II patient both had a C to T change at position 1198 (C1198T) in exon 6. However, this change does not alter the amino acid (L261L) and was also observed in a familial SHFM3 patient linked to chromosome 10q24. It is likely this substitution is a polymorphism. The other polymorphic changes found in patients and one control were an A to T change in intron 8 at position −22 (IVS8–22A>T) and a G to C change in intron 10 at positions +40 and +41 (IVS10+40G>C, IVS10+41G>C).

DISCUSSION

We detected seven missense mutations in P63 in seven out of 10 patients with EEC syndrome. The mutations involved codons for arginine residues at positions 204 (R204Q, R204L), 279 (R279H), and 304 (two R304Q, R304W, R304P). R204, R279, and R304 are considered three of the five mutational hot spots in P63²⁷ and account for 65% (34/52) of all the P63 mutations found in patients with EEC syndrome^11,27,28 (present data).

In the three EEC patients without mutations in the DNA binding of P63, we extended the analysis to the entire coding region and flanking intronic sequences but no mutations were detected. Interestingly, these three patients also had abnormalities of the central nervous system. One patient had hydrocephalus along with typical split foot malformations but atypical changes in the hands (missing thumb, 3–4 syndactyly). This patient also had thin hair, lacrimal duct stenosis, and hypoplastic nipples. The other two patients had typical EEC features in association with colobomatous microphthalmia and microcephaly. It is possible that a large deletion or inversion may have disrupted P63 and another nearby gene involved in the development of the central nervous system (CNS). This might be the case in the two patients with eye defects since a locus for eye formation was mapped to 3q27.³²

The lack of other reported patients with hydrocephalus and SHFM makes it difficult to propose an alternative candidate locus for this phenotype. However, several other loci have been associated with phenotypes including EEC-like features and eye defects. Microcephaly with and without colobomas has been observed in several patients with syndromic SHFM and visible deletions or rearrangements involving the SHFM1 locus on 7q21.3-q22.1.⁷ Thus, it is possible that these three patients could have alterations at this locus. However, no cytogenetically visible rearrangements were detected in these patients.

Deletions of chromosome 2q31–33 have also been observed in patients with SHFM and multiple anomalies including cleft palate, growth retardation, microcephaly, retinal colobomata, and microphthalmia.^13,34 These observations suggest that another locus for an EEC-like syndrome associated with microcephaly, coloboma, and microphthalmia is located on 2q31–33.

One patient with SHFM, cleft lip and palate, microphthalmia, and mental retardation was previously reported.³⁵ This patient carried a chromosome 6;13 translocation, suggesting another possible locus for this condition on either 6q21 or 13q12.³⁵

Since both of our patients with eye defects and EEC features were female, they could represent variants of the X linked male lethal Goltz-Gorlin (focal dermal hypoplasia) syndrome (MIM 305600). This condition shares clinical features with EEC syndrome and also includes a/microphthalmia and coloboma. However, our patients did not have the skin anomalies characteristic of this syndrome.³³

Two of the missense mutations observed in our study (R204L and R304P) have not been described previously. Patient RG, with the R204L mutation, has EEC syndrome without cleft lip/palate (fig 1A). R204W and R204Q substitutions have been reported, and both substitute polar amino acids for arginine, a basic amino acid. It has been suggested that these substitutions create extensive structural rearrangements affecting the DNA binding activity of P63.¹¹ Leucine is also a polar amino acid, and a substitution of leucine for arginine is likely to have a similar effect on the DNA binding activity of P63.

Patient SH, with the R304P mutation, has EEC syndrome associated with mental retardation, blindness, and a seizure disorder (fig 1B). Proline contains a cyclic ring, which creates a fixed kink in a polypeptide chain. Therefore, substitution of proline for arginine should cause a more drastic effect on the DNA binding activity of P63 than the other R304 mutations reported (R304Q, R304W). While our finding raises the possibility of a relationship between this mutation and the patient’s mental retardation, blindness, and seizures, these findings are more likely attributable to an episode of meningitis that she experienced at the age of 10 months. Identification of additional patients with the same mutation would thus be helpful to confirm or exclude a possible role of P63 in brain development and function.

Using SSCP analysis, we have screened 22 non-syndromic SHFM patients and two syndromic SHFM patients for mutations in P63. Sixteen of those with non-syndromic SHFM had long bone deficiency and six had isolated type I SHFM. The other two patients with syndromic SHFM included one with nystagmus (Karsch-Neugebauer syndrome) and one with seizures and mental retardation. No mutations were detected in the entire coding region and intronic flanking sequences of P63 in our panel of 24 SHFM patients.

In two previous studies, sequencing of the entire coding region and the flanking intronic sequences of P63 showed mutations in 16% (6/37) of non-syndromic SHFM cases studied, including one family with evidence of long bone deficiency.^27,28 There are several possible explanations for the discrepancy between these results and our findings. The reported non-syndromic SHFM patients with P63 mutations could represent EEC patients expressing mild ectodermal features. However, extensive clinical investigation in two cases did not show ectodermal defects or clefting that would suggest EEC syndrome.²⁸ Detailed clinical information was not available for the other four non-syndromic SHFM patients.²⁷ Moreover, five out of the six mutations found in these cases differed from those detected in patients with EEC syndrome.

In our study population, 73% (16/22) of non-syndromic SHFM patients had associated long bone deficiency. Previous studies have screened P63 in 37 non-syndromic SHFM patients. However, it is not clear how many of these patients had long bone involvement.^27,28 Mutations in P63 may only be present in a small number of patients with non-syndromic SHFM, most of whom do not have long bone deficiency. This would potentially explain why no mutations were found in our non-syndromic SHFM patients. It is also possible that gross DNA rearrangements (insertions, deletions, or inversions) may exist in these patients, and neither SSCP analysis nor direct sequencing of the P63 coding region would have detected these rearrangements. Alternatively, since several loci have been characterised for isolated SHFM,^5–13 our data support the hypothesis that P63 is not a major cause of non-syndromic SHFM.