A novel phenotypic pattern in X-linked inheritance: craniofrontonasal syndrome maps to Xp22
A novel phenotypic pattern in X-linked inheritance: craniofrontonasal syndrome maps to Xp22George J. Feldman1, Deeann E. Ward1, Elisabeth Lajeunie-Renier4, Dolores Saavedra5, Nathaniel H. Robin1,+, Virginia Proud6, Laura J. Robb7, Vazken Der Kaloustian7, John C. Carey8, M. Michael Cohen, Jr9, Valerie Cormier4, Arnold Munnich4, Elaine H. Zackai1,2, Andrew O. M. Wilkie10, R. Arlen Price3 and Maximilian Muenke1,2,*
1The Children's Hospital of Philadelphia, Division of Human Genetics and Molecular Biology, and Departments of Pediatrics, 2Genetics and 3Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4399, USA, 4Hopital des Enfants-Malades, 75743 Paris Cedex, France, 5Hospital General `Dr Manuel Gea González', Mexico City, Mexico, 6University of Alabama, Laboratory of Medical Genetics, Birmingham, AL 35294, USA, 7The Montreal Children's Hospital, McGill University, Montréal, Québec H3H 1P3, Canada, 8University of Utah, Salt Lake City, UT 84132, USA, 9Department of Oral and Maxillofacial Sciences, Faculty of Dentistry, and Department of Pediatrics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada and 10Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9D5, UK
Received June 11, 1997;Revised and Accepted July 18, 1997
Craniofrontonasal syndrome (CFNS, OMIM 304110) is a distinctive genetic disorder whose main clinical manifestations include coronal synostosis, widely spaced eyes, clefting of the nasal tip and various skeletal anomalies. CFNS originally was thought to be transmitted as an autosomal dominant trait, but recent studies suggest that it is X-linked dominant, whereby all daughters of males are affected, whereas none of their sons are affected. Here we report data confirming that CFNS is X-linked, mapping to a 13 cM interval in Xp22 with a maximum two-point lod score of 3.9 ([theta] = 0) at DXS8022 and a multipoint lod score of 5.08 at DXS1224. Detailed phenotypic analysis shows that females are more severely affected than males, a highly unusual characteristic for an X-linked disorder. CFNS represents the first multiple congenital anomaly syndrome with this unusual phenotypic pattern of X-linked inheritance.
Craniosynostosis is a common birth defect (1 in 2100-2500 infants) which affects the premature fusion of one or more of the cranial sutures and leads to an abnormal head shape. Classic craniosynostosis syndromes such as Apert, Pfeiffer, Crouzon, Jackson-Weiss and Saethre-Chotzen syndromes previously were classified based on their clinical findings and have now been defined at a molecular level (for reviews see 1 ,2 ). The most common craniosynostosis syndrome for which the gene has not been identified is craniofrontonasal syndrome (CFNS, OMIM 304110, ref. 3 ). It was first described in a mother and daughter who both had coronal synostosis with brachycephaly, hypertelorism, downslanting palpebral fissures, clefting of the nasal tip and various digital and joint anomalies (4 ). The inheritance of CFNS has been unclear: while the pedigrees are compatible with both X-linked dominant and autosomal dominant inheritance, the absence of a substantiated instance of male-to-male transmission supports X-linked inheritance. However, expression of CFNS is more severe in females than males-a phenotypic pattern not usually seen in this mode of inheritance (5 ,6 ). Based on a female with CFNS and a chromosomal deletion involving the terminal region of Xp (7 ), the gene map locus for CFNS was tentatively assigned to Xp22 (3 ). To test this hypothesis, we used linkage analysis in 12 CFNS families and were able to map the putative CFNS gene to a 13 cM interval in Xp22. Phenotypic analysis in these families showed that females were more severely affected than males, which is in contrast to either X-linked recessive or dominant inheritance. To our knowledge, CFNS represents the first craniofacial anomaly with this novel phenotypic pattern of X-linked inheritance.
As an initial step toward defining the molecular basis for CFNS, we conducted linkage analysis in 12 unrelated CFNS families with two or more affected family members in 2-4 generation families (Fig. 1 ). In these families, there are 15 affected males who inherited the mutant gene from their mothers; six unaffected males were born to carrier mothers. Four of the affected males have had children of whom seven are daughters, all of whom are affected, and three sons are unaffected, consistent with X-linked inheritance. Representative affected individuals are shown from kindreds 1, 6, 12 and 13 (Fig. 2 ). In our linkage analysis, we concentrated our initial efforts on Xp markers based on a report of a female with CFNS and deletion of Xp22 (7 ). We tested 14 polymorphic microsatellite markers from Xp: Xpter-DXS1060-(DXS1223-DXS9036)-DXS1043-DXS1224- DXS8022-DXS8108-DXS1053-DXS987-DXS999-DXS1226-DXS989-DXS1214-DXS1068-Xcen. The maximum two-point lod score of 3.9 was found with DXS8022 at zero recombination. Crossover events were noted for marker DXS9036 in individual II-3 in family 6, and marker DXS1053 in individual II-3 in family 5. These crossovers allow us to place the putative CFNS gene in a 13 cM interval bordered distally by DXS9036 and proximally by DXS1053. Based on an integrated yeast artificial chromosome (YAC) map of the human X chromosome, this interval is estimated to be ~8.7 Mb (Whitehead map and ref. 8 ). Multipoint linkage analyses result in a multipoint lodscore of 5.08 for DXS1224 (Fig. 3 , Table 1 ).
In the present study, we included phenotypic data on individuals only when sufficient clinical and molecular information was available. On detailed clinical analysis (Table 2 ), the majority of males were mildly affected, with hypertelorism as their only sign, and none had coronal synostosis, in contrast to the findings in their female relatives (Fig. 2 ). In females, findings include severe hypertelorism with extremely broad nasal root, and severe craniofacial asymmetry including orbital asymmetry probably caused by unicoronal synostosis (Fig. 2 ). With rare exceptions (i.e. individual I-2 in family 1, who is phenotypically normal) females were more severely affected than males. In addition, five individuals had uni- or bilateral cleft lip and/or palate, consistent with the suggestion that cleft lip and/or palate is an associated feature in CFNS (9 ).
The clinical findings in CFNS initially were described in a mother and daughter with ocular hypertelorism and coronal synostosis (4 ). In a review of 18 CFNS families with 66 affected individuals, females had hypertelorism, broad nasal root, frontal bossing, craniosynostosis, syndactyly of the fingers and toes and vertical grooving of the nails (6 ). Although males demonstrated many of these signs, they were less severely affected compared with the affected females. In addition, affected males did not have craniosynostosis. X-linked dominant inheritance of CFNS was suggested, based on the documentation of eight affected men who transmitted this condition to all of their 21 daughters but to none of their eight sons (10 ). In contrast to most other known X-linked recessive or dominant disorders, where males are more severely affected than females, the milder phenotype of the males is unexplained.
There are several possible explanations for this paradoxical phenotypic pattern of X-linked inheritance with more severely affected females than males. (i) `Metabolic interference' in which the interaction of a normal and a mutant allele in a multimeric protein produces more severe dysfunction than the mutant allele alone (11 ,12 ). For example, Abruptex (Abx) mutations of the DrosophilaNotch gene fall into two genetic types, enhancers and suppressors of Notch. Homozygotes of either type are viable, yet compound enhancer/suppressor heterozygotes are lethal (13 ,14 ). (ii) A functional homologue on the Y chromosome may ameliorate the effect of the X chromosome mutation. For example, it has been suggested that haploinsufficiency for the X-encoded RPS4X gene, which has a functional Y-encoded homologue RPS4Y, contributes to features of Turner (45,X) syndrome (15 ). (iii) A disturbance in the process of X inactivation of the mutant gene could create a condition of functional disomy in females. Males would be unaffected by this process, as their X chromosome does not undergo inactivation. (iv) CFNS might be a sex-limited disorder, in which the greater severity in females is explained by different interaction of the mutant gene with sex-specific developmental pathways. For example, mutations of HOXA13, an autosomal gene, cause severe uterine abnormalities in females but only hypospadias in males (16 ). However, this hypothesis appears least likely as the developmental defects in CFNS do not seem to involve pathways of sexual differentiation.
Each hypothesis implies a different function for the biology of the normal CFNS gene on the X chromosome. Metabolic interference requires that the gene does not undergo X inactivation and that no functional Y homologue exists, whereas the converse is true for the Y homologue theory. Functional disomy requires that the gene is normally X inactivated, but that no Y homologue exists. Although the first two hypotheses are in contrast to the tendency for genes that escape X inactivation to have Y homologues (and vice versa), thus maintaining dosage compensation, this rule is not absolute (17 ).
Twelve families segregating CFNS participated in this study. Detailed clinical findings had been reported in five of them: kindreds 4 (corresponds to family E in the clinical report), 5 (C), 6 (D), 8 (B) (ref. 5 ) and 10 (ref. 19 ). All participants were clinically re-evaluated by one or more of the authors and their disease status ascertained before molecular studies were initiated. At the time of this re-evaluation, informed consent was obtained in accordance with the standards set by local institutional review boards. Medical records, clinical photographs and autoradiograms were reviewed when available. Clinical findings in affected individuals (12 affected males and 28 affected females) who participated in this study have been summarized in Table 2 . Affected individuals who did not participate in the molecular study or on whom complete clinical information was unavailable were not included.
DNA samples were analysed by polymerase chain reaction (PCR) amplification using 1-5 microsatellite markers per reaction by standard protocols. Fourteen X chromosome primer pairs were used for PCR (Research Genetics, Inc.). In a final volume of 10 [mu]l, 30 ng of genomic DNA was used in 10 mM Tris-HCl, pH 8.4, 1.5 mM MgCl2, 0.01% gelatin, 200 [mu]M each dTTP, dGTP, dATP, 2.5 [mu]M dCTP, 0.35 nCi [[alpha]-32P]dCTP, 1.5 pmol of each primer, and 0.15 U of TaqI polymerase (Perkin Elmer). Thermal cycling (Omnigene) consisted of 27 cycles, with 30 s at 94oC, 75 s at 55oC and 15 s at 72oC. The amplified PCR products were denatured, and separated with size markers on a 6% polyacrylamide gel. The gels were dried and exposed to autoradiography film (Kodak and Fuji). Autoradiograms were recorded independently by two investigators.
The two-point linkage analyses were performed by the computer program MLINK (20 ). The multipoint analyses were completed using LINKMAP (21 ). Both used the FASTLINK algorithm (22 ). The analyses assumed a gene frequency for familial CFNS of 1*10-6, which is similar to frequencies of other rare disorders. Given the confines of the clinical evaluation for some of our subjects, the penetrance of CFNS was estimated as 80% for males inheriting the mutant allele and 90% for females inheriting the mutant allele.
We are grateful to all family members for their participation and help in the study. We thank Dr Laura Cornejo for her help. A.O.M.W. is supported by the Wellcome Trust. This work was supported in part by NIH training grant 5T32HD07107 (to N.H.R.), by NIH grants RO1DK44073 and RO1DK48095 (to R.A.P.), R29HD28732 and RO1HD29862 (to M.M.) and by the Children's Hospital of Philadelphia Development Fund (to M.M.).
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*Present address and address for correspondence: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 10 Center Drive - MSC 1852, Building 10, Room 10-C-101C, Bethesda, MD 20892-1852, USA. Tel: +1 301 402 1159; Fax: +1 301 496 7157; Email: mmuenke@nhgri.nih.gov +Present address: Center for Human Genetics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-6055, USA
S. R. F. Twigg, R. Kan, C. Babbs, E. G. Bochukova, S. P. Robertson, S. A. Wall, G. M. Morriss-Kay, and A. O. M. Wilkie Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome
PNAS,
June 8, 2004;
101(23):
8652 - 8657.
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