Human Molecular Genetics, 2000, Vol. 9, No. 13 1951-1955
© 2000 Oxford University Press
The Conradi–Hünermann–Happle syndrome (CDPX2) and emopamil binding protein: novel mutations, and somatic and gonadal mosaicism
Department of Dermatology, University of Münster, von-Esmarch-Strasse 56, D-48149 Münster, Germany, 1Hospital for Sick Infants, D-09009 Chemnitz, Germany, 2Department of Dermatology, De Heel Hospital, 1500 EE Zaandam, The Netherlands and 3Department of Medical Genetics, University of Manchester, Manchester M13 0JH, UK
Received 22 March 2000; Revised and Accepted 19 June 2000.
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ABSTRACT |
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The Conradi–Hünermann–Happle (CHH) syndrome (X-chromosomal dominant chondrodysplasia punctata type II; MIM 302960) is an X-linked dominant disorder that is characterized by ichthyosis, chondrodysplasia punctata, cataracts and short stature. The disease occurs almost exclusively in females and shows increased disease expression in successive generations (anticipation). Recently, causative mutations in the emopamil binding protein (EBP) have been identified. To better appreciate the genetics of this syndrome we analyzed the EBP gene in seven independent families using PCR, conformation-sensitive gel electrophoresis, direct sequencing and restriction enzyme analysis. We found five novel mutations: three nonsense mutations in exon 2 and exon 3 and two frameshift mutations, one deletion in exon 4 and an insertion in exon 5. In two families, known mutations affecting exon 2 were identified. Surprisingly, we failed to detect the mutation in a grandmother exhibiting minor disease symptoms such as sectorial cataract and attribute this to gonadal and somatic mosaicism. Gonadal mosaicism appeared also to be involved in the case of healthy parents having two affected girls, one of whom died due to the disease. We conclude that gonadal mosaicism has to be considered when dealing with seemingly sporadic cases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Conradi–Hünermann–Happle (CHH) syndrome X-chromosomal dominant chondrodysplasia punctata type II (CDPX2; MIM 302960) is an X-linked dominant disease that was fully delineated by Happle between 1977 and 1981 as an X-linked gene defect (1–4). It is characterized by linear ichthyosis, chondrodysplasia punctata, cataracts and short stature.
At birth the skin often exhibits severe erythroderma and marked scaling which is arranged in particular on the back in whorls and swirls and follows the lines of Blaschko. Erythroderma and scaling on the trunk usually resolve after the first few months of life leaving follicular atrophoderma, hypo- and hyper-pigmentations and circumscribed alopecia on the scalp, whereas ichthyosis on arms and legs often remains present during the entire life.
A further hallmark of the syndrome is the asymmetry of the skeletal involvement: punctate calcifications of the epiphyseal regions usually result in an asymmetric shortening of the long bones, sometimes in very severe kyphoscoliosis, facial dysplasia and congenital hip dislocation. However, one case with a symmetrical shortening of the tubular bones has also been reported (5). Unilateral and sectorial cataracts are further typical signs of the disease (6) and like the linear cutaneous and often asymmetric skeletal involvement reflect differences in X-inactivation. Anticipation (i.e. a stepwise increase in disease expression from one generation to the other) is another striking clinical feature (7,8).
The hypothesis of an unstable premutation (8) as well as the implication of a primary peroxisomal defect (9,10) proved to be incorrect when recently Derry et al. (11) and Braverman et al. (12) identified mutations in the gene that encodes the emopamil binding protein (EBP) in several patients. The EBP gene resides on the short arm of the X chromosome at Xp11.22–p11.23 and could be shown to be deficient in the mouse mutant Tattered (Td), as well. It has a dual function: on the one hand it serves as a binding protein for the Ca2+ antagonist emopamil and thus as a high affinity acceptor for several anti-ischemic drugs (13), and on the other hand it also acts as a 
8–7 sterol isomerase (14).
To better appreciate the genetics of the CHH syndrome we analyzed the EBP gene in seven independent families using PCR, conformation-sensitive gel electrophoresis (CSGE), direct sequencing and restriction enzyme analysis and report here on novel mutations, the presence of gonadal mosaicism and severe intrafamilial variation in disease expression that may in part be related to somatic mosaicism.
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RESULTS |
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Clinical findings
Clinical findings are summarized in Table 1. The pedigrees of the families are depicted in Figure 1.
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Mutation analysis
The mutation screening of the EBP gene revealed heteroduplexes in all index patients of the seven families and in four further affected family members suffering from the CHH syndrome (CDPX2). By direct sequencing we could resolve the CSGE changes in all cases and found two mutations [already reported (11,12)] and five novel, nonsense and frameshift mutations. Family 1 is unique in so far as a CSGE modification was noted in the proposita and her mother, but not in the grandmother who exhibited minor symptoms of the disease such as sectorial cataract and short stature (Fig. 2). In families 1 and 2, two different novel mutations affected the same nucleotide at position 333 in exon 3 resulting in both cases in stop codons at position 111. Tyrosine 111 is a highly conserved amino acid in the cytoplasmic linker domain of EBP. In family 1, cytosine 333 was substituted by guanine (TAC
TAG) (Fig. 3) whereas, in family 2, the same cytosine was changed to adenine (TACTAA). These two nonsense mutations act as null alleles. In family 3, both the proband and her mother had a novel nonsense mutation in exon 2 at nucleotide 203 (GA) which changes the codon for tryptophan to a stop codon at position 68. Tryptophan 68 is a conserved amino acid located in transmembrane domain (TMD) 2 of EBP. A previously reported mutation was found in family 4 (Table 2). In the study by Braverman et al. (12), this nucleotide substitution, R147H, was not identified in 212 chromosomes from control individuals by allele-specific oligonucleotide analysis, and therefore is considered a mutation and not a polymorphism. In the index case of family 5, we identified a deletion in exon 4 at nucleotide 390 (delA) (Fig. 4a) which causes a frameshift and thus results in a premature stop codon at position 137. This mutation could be confirmed by digestion with the restriction endonuclease HaeIII (Fig. 4b). In family 6, we found an insertion in exon 5 at nucleotide 586 (insA) which has similar consequences as the latter mutation, generating a premature termination of the translation. In the proband of family 7, we identified a nonsense mutation, namely R63X at nucleotide 187 (CT), which was reported by Derry et al. (11) and Braverman et al. (12) in their patients. It was suggested to be a hot spot for mutations (11). The results of the mutation analysis and the predicted consequences at the protein level are summarized in Table 2.
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DISCUSSION |
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The genetics of the CHH syndrome (CDPX2) has perplexed medical geneticists, pediatricians and dermatologists for quite some time. Our results extend the recent findings by Braverman et al. (12) and Derry et al. (11) and further establish EBP as the molecule that is deficient in the CHH syndrome. It is of interest that a mutation in the same gene also underlies the mouse mutant Td which is very similar to the mouse mutant bare patches (Bpa). This similarity can now be understood as the mutation underlying Bpa also affects the sterol biosynthesis pathway and results in a similar metabolic block (15). As shown by Moebius et al. (16) amino acids within TMDs 2, 3 and 4 as well as from the cytoplasmic linker domain are essential for enzymatic activity and for binding the ligand emopamil. Our results, similar to those of Braverman et al. (12) and Derry et al. (11), show that mutations occurring in the TMDs are also seen in patients with the CHH syndrome and thus the TMDs also appear to be relevant for the function of EBP as a sterol isomerase. The molecular pathology of the CHH syndrome can certainly not be explained by the anti-ischemic effect of emopamil, but rather by its function in the sterol biosynthesis pathway.
A similar metabolic block has been identified recently in the Smith–Lemli–Opitz syndrome (17) which is caused by a defect in the final enzyme of cholesterol biosynthesis, namely in the gene encoding 7-dehydrocholesterol reductase. In this context, it is noteworthy that the Smith–Lemli–Opitz syndrome shares involvement of chondrodysplasia punctata with the CHH syndrome, and it has been suggested that the skeletal manifestations in both syndromes may derive from accumulation of toxic sterol intermediates that may interfere with the function of cholesterol-modified hedgehog proteins (18,19).
A striking clinical feature of the CHH syndrome is its strong intrafamilial variation which means that the phenotypic effect of a given mutation cannot be predicted. As can be seen in families 1 and 4, one affected girl died shortly after birth whereas another affected sister in both families showed a much milder disease. This variation of disease expressivity within the same mutation and the same family is of paramount importance when providing genetic counselling to CHH syndrome families. We attribute intrafamilial variation occurring in the same generation to differences in Lyonization which certainly also accounts for the marked asymmetry of skeletal and ocular involvement.
Apart from variation affecting sibs in the same generation, stepwise increases in disease expression from one generation to the other are also observed. In the past this phenomenon of anticipation has been explained by the possible involvement of an unstable premutation (8). As we know now, the mutations affecting the EBP gene are not unstable and do not involve trinucleotide repeats. However, in two of the families, we have evidence for the presence of gonadal mosaicism in a parent. In our previous linkage study (8) the grandmother in family 1 was classified as suffering from the CHH syndrome as she exhibited pathognomonic signs of the disease such as sectorial cataract and short stature. It is of interest that we were not able to detect in her the mutation that was present in an affected daughter and granddaughter. Therefore, on the basis of our molecular studies, the affected status of this grandmother has now to be redefined. The most likely explanation that would account both for minor disease expression and for presence of a much more severe disease in her affected offspring is the presence of gonadal and somatic mosaicism in her. Somatic mosaicism in the CHH syndrome has previously been noted in a male patient reported by Metzenberg et al. (20). Gonadal mosaicism in a parent is the most likely explanation for the finding of healthy parents who have two affected children (Fig. 1d, family 4).
In conclusion, a number of the perplexing features of the genetics of the CHH syndrome have now been resolved: (i) the disease is caused by mutations in the gene for EBP; (ii) these mutations are simple substitutions, deletions or insertions and thus are not ‘unstable’; (iii) they can be found in regions that are relevant for the function of EBP; and (iv) clinically unaffected mothers or mothers with very mild disease expression may suffer from somatic mosaicism. Somatic mosaicism can in part explain the phenomenon of anticipation, but differences in X-inactivation may also play a role.
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MATERIALS AND METHODS |
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Family studies
We studied seven families, 25 individuals altogether. Clinical findings for families 1 and 6 have been reported in detail previously (8,21). In five families all patients and further family members were examined by experienced dermatologists (H.T. and E.F.), and two British families were examined by an experienced medical geneticist (D.D.). In addition, one of us (H.T.) carefully reviewed medical records for all families. The pedigrees of the families are shown together with molecular findings in the respective figures. The clinical diagnosis of the CHH syndrome could be made in 11 women.
DNA studies
DNA was isolated from EDTA blood samples using the QIAamp Blood minikit (Qiagen, Hilden, Germany). The four coding exons 2, 3, 4 and 5 of the EBP gene were amplified by PCR using the following intronic primers; for exon 2: 5'-CTTCCTGCCTATACACACGC-3' (forward) and 5'-AGCAAATCCCATCCCACAGC-3' (reverse); for exons 3 and 4: 5'-GTGTGTGTTCCTTTCACTGC-3' (forward) and 5'-CATCTGTGTCTGTGGATCCC-3' (reverse); and for exon 5: 5'-AAGGTGTGAGCTCTCCTGAG-3' (forward) and 5'-GACTAGACTCTTCTGGCAGG-3' (reverse). Cycling conditions were as follows: 40 cycles at 95°C for 5 min, 95°C for 45 s, 54°C for 45 s and 72°C for 45 s and extension at 72°C for 10 min (PE Biosystems, Weiterstadt, Germany). To screen the PCR products for possible mutations, heteroduplex analysis using CSGE was performed (22). PCR products showing conformation changes were then sequenced from both directions on an automated sequencer (Genome Express, Grenoble, France). When possible, the mutations identified were confirmed by digestion with restriction endonucleases. Since the W68X mutation creates a new restriction site for MaeI in exon 2, this enzyme was used to confirm the mutation. The PCR product was incubated for 2 h with MaeI at 45°C and then analyzed on a 3% agarose gel. Accordingly the substitution at nucleotide 333 (CG) which creates a new restriction site for BstYI in exon 3 was analyzed by digestion for 2 h at 60°C. The deletion at nucleotide 390 (delA) which creates a new restriction site for HaeIII was analyzed using this enzyme and incubated at 37°C for 2 h. For the latter two digestions, the products were analyzed on a 3% agarose/TAE gel.
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ACKNOWLEDGEMENT |
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We would like to thank Priv. Doz. Dr Chr Franz, St Marien-Hospital, 53115 Bonn, Germany, for referring the index case of family 2.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 251 8356501; Fax: +49 251 8356945; Email: traupeh@uni-muenster.de
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REFERENCES |
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