DOI: 10.1002/pd.4591

ORIGINAL ARTICLE

Severe X-linked chondrodysplasia punctata in nine new female fetuses Mathilde Lefebvre1,2,3, Fabienne Dufernez4, Ange-Line Bruel2, Marie Gonzales5, Bernard Aral6, Judith Saint-Onge6, Nadège Gigot6, Julie Desir7, Caroline Daelemans8, Frédérique Jossic9, Sébastien Schmitt10, Raphaele Mangione11, Fanny Pelluard12, Catherine Vincent-Delorme13, Jean-Marc Labaune14, Nicole Bigi15, Dominique D’Olne16, Anne-Lise Delezoide17, Annick Toutain18, Sophie Blesson18, Valérie Cormier-Daire19, Julien Thevenon2, Salima El Chehadeh1,2, Alice Masurel-Paulet1, Nicole Joyé5, Claude Vibert-Guigue20, Luc Rigonnot21, Thierry Rousseau22, Pierre Vabres2,23, Philippe Hervé24, Antonin Lamazière25, Jean-Baptiste Rivière2,6, Laurence Faivre1,2, Nicole Laurent3 and Christel Thauvin-Robinet1,2* 1

Centre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l’Est, FHU-TRANSLAD, CHU Dijon, France GAD: EA4271 « Génétique des Anomalies du Développement » (GAD), FHU-TRANSLAD, Université de Bourgogne, Dijon, France 3 Service d’Anatomo-Pathologie, Faculté de Médecine de Dijon, Dijon, France 4 APHP, Hôpital Saint-Antoine, Biochimie B, Laboratoire de Référence pour le Diagnostic Génétique des Maladies Rares, Paris, France 5 Service de Génétique et d’Embryologie Médicales, Université Paris VI, Hôpital Trousseau, Paris, France 6 Laboratoire de Génétique Moléculaire, CHU Dijon, France 7 Center for Medical Genetics, Hospital Erasme, ULB, Brussels, Belgium 8 Institut de Recherches Interdisciplinaires en Biologie Humaine et Moléculaire, Hôpital Erasme, Université Libre de Bruxelles, Brussel, Belgium 9 CHU Nantes, Laboratoire d’anatomopathologie A, Nantes, France 10 CHU Nantes, Service de Génétique Médicale, Nantes, France 11 Department of Gynecology, Hôpital Pellegrin, Bordeaux, France 12 Service de pathologie, CHU de Bordeaux, Bordeaux, France 13 Service de Génétique Médicale, Hôpital Jeanne de Flandre, CHRU de Lille, Lille, France 14 Hôpital de la Croix Rousse, CHU Lyon, France 15 Génétique médicale, CHRU Arnaud de Villeneuve, Montpellier, France 16 Pathological Anatomy and Cytology Centre, Brussels, Belgium 17 Department of Developmental Biology, Hôpital Robert Debré, Paris, France 18 Service de Génétique, Centre Hospitalo-Universitaire Tours, Tours, France 19 Institut Imagine, Hôpital Necker Enfants Malades (AP-HP), Paris, France 20 Service de Gynécologie-Obstétrique, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France 21 Service de Gynécologie-Obstétrique, Centre Hospitalier Sud Francilien (CHSF), Corbeil-Essonnes, France 22 Service de Gynécologie, Hôpital Le Bocage, Dijon, France 23 Service de dermatologie, Hôpital Le Bocage, Dijon, France 24 Service de Gynécologie, Centre Hospitalo-Universitaire Tours, Tours, France 25 Laboratory of Mass Spectrometry-APLIPID, Faculté de Médecine Pierre et Marie Curie, ER7-UPMC, Paris, France *Correspondence to: Christel Thauvin-Robinet. E-mail: [email protected] 2

ABSTRACT Objectives Conradi–Hünermann–Happle [X-linked dominant chondrodysplasia punctata 2 (CDPX2)] syndrome is a rare X-linked dominant skeletal dysplasia usually lethal in men while affected women show wide clinical heterogeneity. Different EBP mutations have been reported. Severe female cases have rarely been reported, with only six antenatal presentations. Methods To better characterize the phenotype in female fetuses, we included nine antenatally diagnosed cases of women with EBP mutations. All cases were de novo except for two fetuses with an affected mother and one case of germinal mosaicism. Results The mean age at diagnosis was 22 weeks of gestation. The ultrasound features mainly included bone abnormalities: shortening (8/9 cases) and bowing of the long bones (5/9), punctuate epiphysis (7/9) and an irregular aspect of the spine (5/9). Postnatal X-rays and examination showed ichthyosis (8/9) and epiphyseal stippling (9/9), with frequent asymmetric short and bowed long bones. The X-inactivation pattern of the familial case revealed skewed X-inactivation in the mildly symptomatic mother and random X-inactivation in the severe fetal case. Differently affected skin samples of the same fetus revealed different patterns of X-inactivation.

Prenatal Diagnosis 2015, 35, 1–10

© 2015 John Wiley & Sons, Ltd.

M. Lefebvre et al.

Conclusion Prenatal detection of asymmetric shortening and bowing of the long bones and cartilage stippling should raise the possibility of CPDX2 in female fetuses, especially because the majority of such cases involve de novo mutations. © 2015 John Wiley & Sons, Ltd.

Funding sources: This work was supported by the Regional Council of Burgundy (to C.T-R). Conflicts of interest: None declared

INTRODUCTION Chondrodysplasia punctata is a rare, heterogeneous group of skeletal dysplasia characterized by a disorder of bone mineralization resulting in epiphyseal stippling. Subtypes are divided into the rhizomelic type, with autosomal recessive inheritance and early lethality caused by homozygous or compound heterozygous mutations in the PEX7 gene, and the non-rhizomelic type (Conradi–Hünermann group). The etiology of this subtype is heterogeneous and involves both teratogenic causes, such as vitamin K antagonists, and genetic causes, with different modes of inheritance.1 The so-called Conradi–Hünermann–Happle syndrome (CHH; MIM 302960) or chondrodysplasia punctata type II follows an X-linked dominant inheritance (CDPX2).2 This syndrome is usually lethal early in pregnancy in hemizygous men while affected women exhibit segmentally arranged clinical features.3,4 Strikingly high clinical variability has been described in women, ranging from severe prenatally diagnosed cases to incomplete penetrance.5 When diagnosed in the neonatal period, affected cases present with chondrodysplasia punctate of the epiphyses of the long bones and vertebrae, rhizomelic and often asymmetric shortening of limbs, scoliosis, ocular abnormalities (premature cataract, microphthalmia, and/or microcornea), a distinctive craniofacial appearance, and ectodermal changes. Linear or blotchy scaling ichthyosis in the newborn usually resolves in the first months of life and leaves linear or whorled atrophic patches involving hair follicles, called follicular atrophoderma. Coarse hair with scarring alopecia, occasional flattened or split nails with normal teeth are also characteristic.6,7 Calcifications classically disappear in the first years of life, and patients display normal psychomotor development and short stature. CDPX2 is caused by mutations in the EBP gene. This gene is located on the short arm of the X chromosome and encodes the emopamil-binding protein, which plays a role in cholesterol biosynthesis: 3-β-hydroxysteroid-Δ8-Δ7-isomerase activity catalyzes an intermediate step in the conversion of lanosterol to cholesterol. CDPX2 patients exhibit abnormally elevated levels of cholesterol precursors in plasma and tissues, which can be determined by gas chromatography– mass spectrometry. 8 To date, more than 60 missense or truncating EBP mutations have been reported, without any clear correlation between mutations and the severity of the clinical phenotype.9 To date, only six female fetuses with severe CDPX2 have been reported.10–13 Here, we present the clinical, radiological, biochemical, and molecular characteristics of nine prenatally diagnosed women who presented with fetal ultrasound findings consistent with the diagnosis of EBP–chondrosyplasia Prenatal Diagnosis 2015, 35, 1–10

punctata and discuss the role of the X-inactivation pattern in the severity of the disease.

MATERIAL AND METHODS Patients Retrospectively, we included all French female cases with ultrasound features of CDPX2, in which an EBP mutation was identified after birth or termination of pregnancy. Cases were collected at the main diagnosis laboratory in France over a period of 12 years. For each case, we recorded family history, prenatal ultrasound results, clinical data at birth or fetal autopsy, X-rays, biochemical (8-dehydro-cholesterol and cholest-8(9)en-3β-ol levels) and EBP mutations results.

X-inactivation analysis DNA was extracted from blood samples and/or fetal tissues after informed consent had been obtained. X-inactivation patterns were analyzed by determining the methylation status in exon 1 of the androgen receptor gene (HUMURA) as previously described.14 Briefly, genomic DNA from frozen fetal tissue sample was amplified by polymerase chain reaction (PCR) before and after HpaII digestion. The fluorescently labeled forward primer and unlabeled reverse primer for this reaction were designed from sequences flanking the polymorphic CAG repeat and two methylation-sensitive HpaII sites present in the exon. PCR products were loaded into an autosequencer (Applied Biosystems 3130xl) and examined for marker sizes and peak heights. After compensation for unequal amplification caused by different product sizes and slippages, ratios of inactivation between the two X chromosomes were calculated according to the method of Kubota et al. (1999).14

RNA isolation Total RNA was isolated with the Rneasy Plus Universal Mini kit (Qiagen) according to the manufacturer’s protocol, using TissueLyser II. The integrity of the RNA was assessed on 1.2% agarose gel, and the concentration and purity were determined by optical densitometry. One microgram of total RNA was transcribed into cDNA with the Quantitect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol.

cDNA analysis cDNA synthesis was performed as described earlier. PCR primers were positioned in exon 1 and 5 (design available on request). cDNA was amplified using PrimeStar GXL (Takara) following the instructions provided by the © 2015 John Wiley & Sons, Ltd.

Prenatal Diagnosis 2015, 35, 1–10

+

+

+

Large metaphyses

Epiphyseal stippling

Irregular aspect of the spine

+

2465 g (90th per) +

2190 g (>95th per)

40 cm (50th per)

+

+

+

Asymmetric shortening of the long bones

Bowing of the long bones

Punctuate epiphysis

Radiological features

Others

+

+

Hyperflexion of the left arm Hyperextension of the feet

+

+

+

+

+

+

Bilateral brachymesophalangy of the 5th finger Bilateral clinodactyly Left clubfoot Absence of eyelashes

Hydrops Hypertelorism

Unilateral club foot

+

+

+

Bowed legs

+

+

+

+

591 g (10th per)

28.5 cm ( A/ p.Arg110*

c.469G > C/p.Gly156Arg c.328C > T/p.Arg110*

Severe CDPX2 in female fetuses

WHAT’S ALREADY KNOWN ABOUT THIS TOPIC?

Prenatal Diagnosis 2015, 35, 1–10

WHAT DOES THIS STUDY ADD? *postaxial polydactyly.

Mutation (DNA/protein)

Molecular results

• Chondrodysplasia punctata 2, X-linked dominant (CDPX2) is due to a mutation in EBP. Women presenting with CDPX2 show wide clinical heterogeneity. To date, severe antenatal presentations of CDPX2 have been reported in six female cases.

• This study describes the largest cohort of antenatally diagnosed EBP-related CDPX2 female fetuses including two familial cases. Prenatal detection of asymmetric shortening and bowing of the long bones and cartilage stippling should be CPDX2 in female fetuses, especially because the majority of such cases involve de novo mutations. The high phenotype variability is most probably due to the random X-inactivation.

© 2015 John Wiley & Sons, Ltd.

M. Lefebvre et al.

REFERENCES 1. Hall JG, Pauli RM, Wilson KM. Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 1980;68(1):122–40. 2. Heselson NG, Cremin BJ, Beighton P. Lethal chondrodysplasia punctata. Clin Radiol 1978;29(6):679–84. 3. Aughton DJ, Kelley RI, Metzenberg A, et al. X-linked dominant chondrodysplasia punctata (CDPX2) caused by single gene mosaicism in a male. Am J Med Genet A 2003;116A(3):255–60. 4. Ikegawa S, Ohashi H, Ogata T, et al. Novel and recurrent EBP mutations in X-linked dominant chondrodysplasia punctata. Am J Med Genet 2000;94(4):300–5. 5. Shirahama S, Miyahara A, Kitoh H, et al. Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata. Hum Genet 2003;112(1):78–83. 6. Happle R (1979) X-linked dominant chondrodysplasia punctata. Review of literature and report of a case. Hum Genet 53:65–73. 7. Kolb-Mäurer A, Grzeschik KH, Bröcker EB, Hamm H. Conradi– Hünermann–Happle syndrome (X-linked dominant chondrodysplasia punctata) confirmed by plasma sterol and mutation analysis. Acta Derm Venereol 2008;88:47–51. 8. Braverman N, Lin P, Moebius FF, et al. Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi–Hünermann syndrome. Nat Genet 1999;22(3):291–4. 9. Herman GE, Kelley RL, Pureza V, et al. Characterization of mutations in 22 females with X-linked dominant chondrodysplasia punctata (Happle syndrome). Genet Med 2002;4:434–8. 10. Hellenbroich Y, Grzeschik KH, Krapp M, et al. Reduced penetrance in a family with X-linked dominant chondrodysplasia punctata. Eur J Med Genet 2007;50(5):392–8. 11. Umranikar S, Glanc P, Unger S, et al. X-Linked dominant chondrodysplasia punctata: prenatal diagnosis and autopsy findings. Prenat Diagn 2006;26(13):1235–40. 12. Offiah AC, Mansour S, Jeffrey I, et al. Greenberg dysplasia (HEM) and lethal X linked dominant Conradi–Hünermann chondrodysplasia punctata (CDPX2): presentation of two cases with overlapping phenotype. J Med Genet 2003;40(12):e129. 13. Rakheja D, Read CP, Hull D, et al. A severely affected female infant with x-linked dominant chondrodysplasia punctata: a case report and a brief review of the literature. Pediatr Dev Pathol 2007;10(2):142–8. Review.

Prenatal Diagnosis 2015, 35, 1–10

14. Kubota T. A new assay for the analysis of X-inactivation in carriers with an X-linked disease. Brain Dev 2001;23(Suppl 1):S177–81. 15. Has C, Bruckner-Tuderman L, Müller D, et al. The Conradi– Hünermann–Happle syndrome (CDPX2) and emopamil binding protein: novel mutations, and somatic and gonadal mosaicism. Hum Mol Genet 2000;9(13):1951–5. 16. Kelley RI, Wilcox WG, Smith M, et al. Abnormal sterol metabolism in patients with Conradi–Hünermann–Happle syndrome and sporadic lethal chondrodysplasia punctata. Am J Med Genet 1999;83(3):213–9. 17. Thauvin-Robinet C, Cossée M, Cormier-Daire V, et al. Clinical, molecular, and genotype–phenotype correlation studies from 25 cases of oral-facial-digital syndrome type 1: a French and Belgian collaborative study. J Med Genet 2006;43(1):54–61. 18. Crovato F, Rebora A. Acute skin manifestations of Conradi– Huenermann syndrome in a male adult. Arch Dermatol 1985;121 (8):1064–5. 19. Hochman M, Fee WE Jr. Conradi–Hunerman syndrome. Case report. Ann Otol Rhinol Laryngol 1987;96(5):565–8. 20. De Raeve L, Song M, De Dobbeleer G, et al. Lethal course of X-linked dominant chondrodysplasia punctata in a male newborn. Dermatologica 1989;178(3):167–70. 21. Tronnier M, Froster-Iskenius UG, Schmeller W, et al. X-chromosome dominant chondrodysplasia punctata (Happle) in a boy. Hautarzt 1992;43(4):221–5. 22. Omobono E, Goetsch W. Chondrodysplasia punctata (the Conradi– Hünermann syndrome). A clinical case report and review of the literature. Minerva Pediatr 1993;45(3):117–21. 23. Sutphen R, Amar MJ, Kousseff BG, Toomey KE. XXY male with X-linked dominant chondrodysplasia punctata (Happle syndrome). Am J Med Genet 1995;57(3):489–92. 24. Milunsky JM, Maher TA, Metzenberg AB. Molecular, biochemical, and phenotypic analysis of a hemizygous male with a severe atypical phenotype for X-linked dominant Conradi–Hunermann–Happle syndrome and a mutation in EBP. Am J Med Genet A 2003;116A (3):249–54. 25. Morleo M, Franco B. Dosage compensation of the mammalian X chromosome influences the phenotypic variability of X-linked dominant male-lethal disorders. J Med Genet 2008;45(7):401–8. DOI:10.1136/jmg.2008.058305.

© 2015 John Wiley & Sons, Ltd.