Identification of Causative Mutation in a Korean Family with Crouzon

29 downloads 0 Views 549KB Size Report
Crouzon Syndrome Using Whole Exome Sequencing. Borum Sagong1,2,*, Da .... tory canals, vestibular aqueduct, and middle ear struc- tures were analyzed on ...
Available online at www.annclinlabsci.org

476

Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

Identification of Causative Mutation in a Korean Family with Crouzon Syndrome Using Whole Exome Sequencing Borum Sagong1,2,*, Da Jung Jung3,*, Jeong-In Baek1, Min-A Kim1,2, Jaetae Lee4, Sang-Heun Lee3, Un-Kyung Kim1,2, and Kyu-Yup Lee3 1Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea, 2School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, South Korea, 3Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea, and 4Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, South Korea

Abstract. Craniosynostosis is a heterogeneous disorder that results in a common malformation which causes premature fusion of one or more cranial sutures. Whole-exome sequencing (WES) was recently developed as a powerful genetic strategy for identifying pathogenic mutations of heterogeneous disorders with various causative genes. A 24-year-old woman visited our department for evaluation of persistent hearing impairment and absence of an external auditory canal from birth. In this study, we performed WES to identify the causative mutation in a Korean family who has Crouzon Syndrome (CS). We first focused on 16 genes associated with craniosynostosis and sorted the heterozygous variations according to the autosomal dominant inheritance pattern of her family. After the bioinformatic analysis for filtering and detecting variations, three non-synonymous variations in different genes were selected for additional analysis. Among these, the p.C278F mutation in the FGFR2 gene was only absent from both dbSNP and the 1000 Genomes database. We considered the p.C278F mutation in the FGFR2 gene as the causative mutation for the CS. This result suggests that the application of WES will be valuable for diagnosis of congenital disorders with clinical and genetics heterogeneities. Key words: Crouzon syndrome, fibroblast growth factor, craniosynostosis, FGFR2, whole-exome sequencing. Introduction Craniosynostosis is a common malformation occurring in 1 of 2,500 live births [1]. This condition results in premature fusion of one or more cranial sutures, leading to defective growth and development of the orbital and maxillary complex, which causes shallow orbit, maxillary hypoplasia, and midfacial hypoplasia [2-6]. Approximately 15% of all craniosynostosis cases comprise syndromic craniosynostosis, resulting in more severe conditions than non-syndromic cases [7]. There are more than 180 syndromes that manifest craniosynostosis [1], *These authors contributed equally to this work. Address correspondence to Un-Kyung Kim, Ph.D., Department of of Biology, College of Natural Sciences, Kyungpook National University, Daegu, South Korea; phone: +82 53 950 5353; fax: +82 53 953 3066; e mail: [email protected], or Kyu-Yup Lee, MD, Ph.D., Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, Kyungpook National University, Daegu, South Korea; phone: +82 53 420 5777; fax: +82 53 423 4524; e mail: [email protected]

the most common being Pfeiffer, Crouzon, Apert, and Saethre-Chotzen [3,8]. These syndromes present with characteristic clinical features, including calvarial synostosis [7]. To date, sixteen genes associated with craniosynostosis have been identified. Even though craniosynostosis can be classified into several different syndromes based on its clinical phenotypes, the correlation between genotype and phenotype does not appear to be strong [9-13]. Moreover, genetic causes have been identified in 45% of patients with craniosynostosis to date, suggesting the possibility of novel causative genes [7]. In this study, we conducted whole-exome sequencing (WES) to identify the causative mutation in a Korean family composed of a young female with congenital aural atresia, hypertelorism, and exophthalmos.

0091-7370/14/0400-476. © 2014 by the Association of Clinical Scientists, Inc.

Genetic diagnosis of craniosynostosis using WES

477

Figure 1. Clinical findings of mild hypertelorism, exophthalmos and aural atresia. Frontal (A), lateral (B), and inferior (C) views of the patient in this study. The three dimensional facial computed tomography images of the patient showing hypoplastic maxilla (white arrow, D-F), protruded mandible (red arrow, E and F) and absence of external auditory canal (asterisk, E). The external auditory canal stenosis on right (G) and left sides (H). Normal right (I) and left (J) auricles in the patient.

Materials and Methods Subjects and clinical evaluations. A 24-year-old woman and her family were subjected to detailed clinical evaluation, including family history, medical history, otologic examinations, audiologic testing, and speech evaluation. Written informed consent was obtained from the participants, and this study was approved by the Institutional Review Board of the Kyungpook National University Hospital. Pure tone audiometry (PTA) with air and bone conduction was performed in a sound controlled room at frequencies ranging from 500 to 8000 Hz, according to standard protocols. The temporal bone computed tomography (CT) scan was performed with a 16 multidetector row CT scanner (Somatom Sensation 16; Siemens, Erlangen, Germany) using a standard temporal bone protocol. Contiguous 0.7-mm scans of the temporal bone were acquired in the axial plane and reformatted coronally with 1.0-mm increments. CT images were performed, digitally stored, and displayed by using the Picture Archiving Communication System (PACS) (Centricity; GE Healthcare, Milwaukee, WI). The threedimensional (3D) images created after multidetector CT examination in detecting acute post-traumatic osseous pathology of the skeletal system. The morphologies of the cochlea, vestibule, semicircular canals, internal auditory canals, vestibular aqueduct, and middle ear structures were analyzed on temporal bone CT scans.

Whole exome sequencing. The genomic DNA was extracted from peripheral blood of the patient and her family members using a FlexiGene DNA kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Five microgram genomic DNA of three individuals in the family (II:2, III:3, and III:4) was fragmented, adapters were attached, and the fragments were fractionated by size to approximately 250 base pairs. Target enrichment was performed using NimbleGen’s SeqCap exome library v.2.0 (Roche/NimbleGen, Madison, WI, USA), and the captured target DNA was sequenced using paired-end reads on an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA). At each step, DNA products were qualified using an Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA). All bioinformatic analyses for filtering and detecting variations were performed using the DNAnexus platform (DNAnexus, Inc., Mountain View, CA, USA, http://www.dnanexus. com/). The exome sequencing reads were aligned to the human reference genome hg19 (University of California, Santa Cruz, CA, USA). Quality filtering was performed based on the PHRED score (> 20). The potential candidate variant was verified by co-segregation with the phenotype within this family based on Sanger sequencing. Primers were designed via Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). PCR products were analyzed using the BigDye Terminator v3.1 Cycle Sequencing Kit and the 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

478

Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

of air-conduction level calculated as the average of the threshold measured at 0.5, 1.0, 2.0, and 3.0 kHz was 60 dB and the average of bone-conduction threshold was 18 dB in the right ear (Figure 2A). In the left side, the average of air-conduction threshold was 52 dB and that of bone-conduction threshold was 23 dB (Figure 2B). The audiologic Figure 2. Preoperative and postoperative pure tone audiometry and temporal tests revealed typibone computed tomography (CT) images. The preoperative audiograms show the avercal conductive age of air-conduction level was 60 dB and the average of bone-conduction threshold was 18 hearing loss coincidB in the right ear (A). In the left side, the average of air-conduction threshold was 52 dB and that of bone-conduction threshold was 23 dB (B). The postoperative audiograms show dent with aural that the averages of air-conduction threshold are 37 dB and 35 dB in the right and left ear, atresia. The patient respectively (A and B). Complete obstructed external auditory canal (asterisks) of left (C) was subjected to raand right (D) ears are shown in the axial view of temporal bone CT. After left ear canaloplasty, the axial view of temporal bone CT shows patent ear canal (E). On the operation, diologic investigaanomalous malleus-incus complex (size, 4 X 4 mm) was found (F). It shows fused malleus tions through temhead and incus body, while the handle of malleus and incus short process look intact. poral bone CT. A completely obThe data were analyzed using Sequencing Analysis v5.2 structed EAC was found (Figure 2C-D). Her ap(Applied Biosystems, Foster City, CA, USA) and pearance resembled that of family members, inChromas Lite v2.01 (Technelysium Pty Ltd., Tewantin, cluding her mother, grandfather, one brother, and QLD, Australia) software. two sisters. These individuals all showed hypertelorism and exophthalmos, but she was the only Results one with aural atresia. Her pedigree represented autosomal-dominant transmission (Figure 3). Clinical presentations. A 24-year-old woman visited our department for evaluation of persistent Middle ear surgeries and auditory rehabilitahearing impairment and absence of an external au- tion. As impaired hearing of both ears affected her ditory canal (EAC) from birth. Upon general ex- daily life, left ear canaloplasty had been planned amination, she showed symptoms characteristic of prior to right side. On operation, an atretic bony midfacial hypoplasia and dysmorphic features, in- plate was exposed after drilling the space between cluding mild hypertelorism and mild exophthal- antero-inferiorglenoid fossa, inferior temporal line, mos, but not severe ophthalmologic problems such and posterior mastoid air cell. The fused malleusas dry eye or hypertonia oculi (Figure 1A-C). incus (M-I) complex that was known to be a develHypoplastic maxilla and protruding mandible rep- opmental problem of the first branchial arch was resent a typical appearance of the Crouzon syn- checked and extracted following removal of atretic drome (CS) (Figure 1D-F). Despite the complete bony plate (Figure 2F). The incus separated from absence of EACs on both sides (Figure 1D-H), her the M-I complex extracted was relatively intact, so auricles were of regular size and appeared normal the incus was sculpted and placed on the stapes (Figure 1I-J). Upon PTA, the average head. After temporalis muscle fascia was grafted on

Genetic diagnosis of craniosynostosis using WES

Figure 3. The pedigree and phenotype of a family with Crouzon syndrome. The left-half-shaded symbols indicate hypertelorism and exophthalmos. The right-half-shaded symbols indicate aural atresia. The patient (III:4) had hypertelorism, exophthalmos, and aural atresia. The filled symbols indicate affected individuals, while unaffected individuals are not shaded. The arrow indicates the proband.

it, the skin harvested in the left thigh in advance was also grafted on the newly created canal. Canaloplasty for right aural atresia was performed 10 months after left ear surgery. Follow up audiologic tests at three month after canaloplasty in both ears presented that her hearing improved (Figure 2A-B). Molecular analysis. WES was performed on samples from two affected members (II:2, III:4) and one unaffected member (III:3) of the family. A total of 3.72 to 4.41 gigabases (Gb) of mapped reads were obtained from each individual. To analyze the exonic sequences preferentially in genes previously known to be associated with craniosynostosis, variation filtering was performed using 16 craniosynostosis-associated genes (Table 1). This sequencing covered more than 96% of the 47.7 kilobases (Kb) of exonic sequences in the 16 genes with a mean depth ranging from 47x to 56x. More than 89.7% of the 16 genes were covered by ten or more reads, demonstrating the high quality of the sequencing (Table 2). A total of 71 to 88 variants were detected in each individual. We first focused on heterozygous variants in the coding sequences of the 16 genes. We identified three variants (c.619G>A in RAB23, c.1522A>G in ESCO2, and c.8323G>T in FGFR2) for follow up based on co-segregation of genotype with phenotype in these three family members, and all three variations were non-synonymous variations. The first variation in the RAB23 gene

479

(c.619G>A) was conversion of glycine to serine at amino acid position 207 (p.G207S). The second variation in the ESCO2 gene (c.1522A>G) caused an amino acid change from isoleucine to valine at position 508 (p.I508V). These two variations were present in the 1000 genomes database (http:// www.1000genomes.org) with minor allele frequencies of 10.6% and 2.1%, respectively (rs1040461 for p.G207S and rs114956994 for p.I508V). The last variation in the FGFR2 gene (c.833G>T) converted cysteine to phenylalanine at amino acid position 278 (p.C278F). This variation was absent from both the dbSNP of NCBI (http://www.ncbi.nlm.nih.gov/) and the 1000 genomes database. In addition, mutation was previously reported to cause craniosynostosis [10,14]. We also confirmed the genotypes of family members using Sanger sequencing and found that they matched exactly (Figure 4). Taken together, these findings indicate that the p.C278F variation in the FGFR2 gene is the causative mutation of the disorder. Discussion CS is the most common craniosynostosis syndrome, responsible for approximately 4.8% of craniosynostosis cases [5,15-20]. It is an autosomal-dominant inherited disorder that is associated with hypertelorism, exophthalmos, parrot-beaked nose, and maxillary hypoplasia with relative prognathism [3,4,6]. CS, known as a branchial arch syndrome, affects development of the ear, which is closely related to first pharyngeal arch. The first branchial arch has been regarded the matrix of the auricle, auditory canal, and ossicles. The head of the malleus and body of the incus originate from the first branchial arch, giving CS has a deep relation with malformation of these ossicles. The external auditory meatus comes from a dent in the first branchial cleft, causing possible incomplete occurrence. In the literature, approximately 55% of patients with CS have conductive hearing loss because of an external auditory canal stenosis or aural atresia, including ossicular anomaly, necessitating a hearing aid or surgery, including canaloplasty. The patient of this study had conductive hearing loss due to an absent external auditory tract, in addition to fusion of the malleus head and incus body, in accordance with CS. A newly formed EAC and reconstruction of the ossicular chain that resulted from canaloplasty improved her hearing.

480

Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

Table 1. The causative genes associated with craniosynostosis syndromes. Genes FGFR2 FGFR3 TWIST1 EFNB1 FGFR1 POR RAB23 EFNA4 ESCO2 GLI3 JAG1 KRAS RECQL4 TGFBR1 TGFBR2 MSX2

Disorders

References

Apert, Crouzon, Pfeiffer, Jackson-Weiss, Beare-Stevenson, and Saethre-Chotzen syndromes Muenke and Crouzon syndromes Saethre-Chotzen syndrome Pfeiffer and Craniofrontonasal syndromes Mild Pfeiffer syndrome Antley-Bixler syndrome Carpenter syndrome Non-syndromic coronal synostosis Roberts syndrome Greig cephalopolysyndactyly Alagille syndrome Noonan syndrome Baller-Gerold syndrome Loeys-Dietz syndrome Loeys-Dietz syndrome Boston-type craniosynostosis

[13, 30-32] [33, 34] [35, 36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [47] [48]

Table 2. Run statistics and coverage of exome-sequencing in genes previously known to be associated with craniosynostosis. Subject

Total mapped reads (Gbp)

Covered target sequence (bp)

Target coverage (%)

Mean depth of coverage (x)

Depth ≥10x of coverage (%)

II:2 III:3 III:4

4.25 3.72 4.41

46,289 45,897 46,578

97.11 96.28 97.71

56.93 47.19 55.36

91.61 89.68 92.26

The radiologic findings and distinguishing clinical features such as exophthalmos, hypertelorism, maxillary hypoplasia, and occasional aural atresia aided in diagnosis. In addition to these presentations, cervical spine abnormalities (30%) and acanthosis nigricans have been noted [3,4,6,21]. Although an increased risk of hydrocephalus (30%) is known, most patients (97%) have normal intelligence [22,23]. For reliable diagnosis of CS, careful evaluation of family history and genetic testing is recommended [21]. WES may revolutionize clinical diagnostics by enabling large parallel identifications of mutations causing a genetic disorder. Although traditional Sanger sequencing based gene specific approaches have led to great insights into inherited disorders over the past few decades, they are unable to detect genetic variations widely and simultaneously [24,25]. A next generation sequencing technique has enabled investigators to obtain variant information down to single-base resolution in a

high-throughput fashion on the scale of the whole human genome [25]. The capacity to screen many genes simultaneously makes this technique an especially powerful tool for detecting pathogenic mutations that cause heterogeneous disorders such as craniosynostosis. A number of recently published studies have successfully employed massively parallel sequencing for heterogeneous disorder analysis [26,27]. Therefore, mutations in genes that are known to cause disorders will also be identified frequently when WES is applied to genetically heterogeneous disorders that can be caused by monogenic mutations in many different genes. In this study, WES was used to analyze a small Korean small family with craniosynostosis for the first time, leading to identification of the causative mutation of this family in the FGFR2 gene. In craniosynostosis, the FGFR2 gene is mutated in Apert, Crouzon, Pfeiffer, Jackson-Weiss, BeareStevenson, and Saethre-Chotzen syndromes (Table 1). However, causative mutations identified in the

Genetic diagnosis of craniosynostosis using WES

Figure 4. The identification of p.C278F mutation in the FGFR2 gene showing co-segregation with Crouzon syndrome. Top-left panel: visualization of affected individual sequencing reads covering the mutation in the FGFR2 gene. Top-right panel: visualization of unaffected individual sequencing reads covering the mutation in the FGFR2 gene. Blue and green reads represent the positive and negative strands, respectively. Red bases represent bases that differ from the black reference sequence. Bottom panel: verification of p.C278F mutation in family individuals by Sanger sequencing. The mutations marked by black arrows are single nucleotide substitutions leading to a change in the amino acid.

same gene differ depending on syndrome. To date, more than 50 mutations have been reported in CS [28]. In the p.C278F mutation identified in this study, alteration of the cysteine at amino acid position 278 affects the ability to form a disulfide bond with the cysteine at amino acid position 342, maintaining the secondary structure of the third Ig-like domain [10,14]. This mutation has been reported in patients with Pfeiffer syndrome and CS [10,14,28]. Similarly, the same mutation has been identified in patients with different syndromes, making the genotype-phenotype correlation unclear [28,29]. Thus, further studies of the other factors in the pathway associated with FGFR2 are needed.

481

Craniosynostosis has shown clinical and genetic heterogeneity through a number of previous studies. These findings indicate that accurate diagnosis of the syndromes associated with craniosynostosis requires comprehensive analysis, including clinical characterization and genetic analysis. In this study, we performed the clinical evaluation of a patient with craniosynostosis using various clinical tests and mutational analysis with WES, providing strong genetic evidence to support this clinical diagnosis. This study suggested the potential to advance genetic counseling and early diagnosis of craniosynostosis through database construction, using WES as a powerful genetic tool of hereditary disorders that have various causative genes and difficult clinical diagnosis. This study also showed that proper clinical intervention is necessary and beneficial for the patients with CS when considering hearing and speech development.

Acknowledgements This work was supported by a grant of the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111774 to K.Y.L., A111345 to U.K.K.). References 1. 2. 3. 4. 5.

Kimonis, V., Gold, J.A., Hoffman, T.L., Panchal, J., and Boyadjiev, S.A. Genetics of craniosynostosis. Semin Pediatr Neurol 2007;14:150-61. Rice, D.P. Clinical features of syndromic craniosynostosis. Front Oral Biol 2008;12:91-106. Babic, G.S. and Babic, R.R. Opthalmological and radiological picture of crouzon syndrome: A case report. Acta Medica Medianae 2009;48:37-40. Kaur, H., Singh Waraich, H., and Sharma, C.M. Crouzon syndrome: A case report and review of literature. Indian J Otolaryngol Head Neck Surg 2006;58:381-2. Pournima, G., Monica, Y., and Meghna, S. Crouzon syndrome: A case report. European J Dent Med 2011;10:1-5.

482 6.

Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

Rani, P.J., Shailaja, S., Srilatha, S., Sridevi, K., Payal, and Vinod, V.C. Crouzon syndrome: A case report. Int J Dent Case Rep 2012;2:117-22. 7. Agochukwu, N.B., Solomon, B.D., and Muenke, M. Impact of genetics on the diagnosis and clinical management of syndromic craniosynostoses. Childs Nerv Syst 2012;28:1447-63. 8. Khan, S.H., Nischal, K.K., Dean, F., Hayward, R.D., and Walker, J. Visual outcomes and amblyogenic risk factors in craniosynostotic syndromes: a review of 141 cases. Br J Ophthalmol 2003;87:999-1003. 9. Baroni, T., Carinci, P., Lilli, C., Bellucci, C., Aisa, M.C., Scapoli, L., Volinia, S., Carinci, F., Pezzetti, F., Calvitti, M., Farina, A., Conte, C., and Bodo, M. P253R fibroblast growth factor receptor-2 mutation induces RUNX2 transcript variants and calvarial osteoblast differentiation. J Cell Physiol 2005;202:524-35. 10. Meyers, G.A., Day, D., Goldberg, R., Daentl, D.L., Przylepa, K.A., Abrams, L.J., Graham, J.M., Jr., Feingold, M., Moeschler, J.B., Rawnsley, E., Scott, A.F., and Jabs, E.W. FGFR2 exon IIIa and IIIc mutations in Crouzon, Jackson-Weiss, and Pfeiffer syndromes: evidence for missense changes, insertions, and a deletion due to alternative RNA splicing. Am J Hum Genet 1996;58:491-8. 11. Park, J., Park, O.J., Yoon, W.J., Kim, H.J., Choi, K.Y., Cho, T.J., and Ryoo, H.M. Functional characterization of a novel FGFR2 mutation, E731K, in craniosynostosis. J Cell Biochem 2012;113:457-64. 12. White, K.E., Cabral, J.M., Davis, S.I., Fishburn, T., Evans, W.E., Ichikawa, S., Fields, J., Yu, X., Shaw, N.J., McLellan, N.J., McKeown, C., Fitzpatrick, D., Yu, K., Ornitz, D.M., and Econs, M.J. Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation. Am J Hum Genet 2005;76:361-7. 13. Wilkie, A.O., Slaney, S.F., Oldridge, M., Poole, M.D., Ashworth, G.J., Hockley, A.D., Hayward, R.D., David, D.J., Pulleyn, L.J., Rutland, P., Malcolm, S., Winter, R.M., and Reardon, W. Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet 1995;9:165-72. 14. Oldridge, M., Wilkie, A.O., Slaney, S.F., Poole, M.D., Pulleyn, L.J., Rutland, P., Hockley, A.D., Wake, M.J., Goldin, J.H., Winter, R.M., Reardon, W., and Malcolm, S. Mutations in the third immunoglobulin domain of the fibroblast growth factor receptor-2 gene in Crouzon syndrome. Hum Mol Genet 1995;4:1077-82. 15. Ahmed, I. and Afzal, A. Diagnosis and evaluation of Crouzon syndrome. J Coll Physicians Surg Pak 2009;19:318-20. 16. Horbelt, C.V. Physical and oral characteristics of Crouzon syndrome, Apert syndrome, and Pierre Robin sequence. Gen Dent 2008;56:132-4. 17. Locuratolo, N., Baffico, M., Baldi, M., Parisi, V., Micacchi, F., Angelucci, V., Rojas Beccaglia, M., Pirro, C., and Fattapposta, F. A novel fibroblast growth factor receptor 2 (FGFR2) mutation associated with a mild Crouzon syndrome. Arch Ital Biol 2011;149:313-7. 18. Murano, I. [Crouzon syndrome]. Nihon Rinsho 2006;Suppl 3:416-7. 19. Singer, S.L., Walpole, I., Brogan, W.F., and Goldblatt, J. Dentofacial features of a family with Crouzon syndrome. Case reports. Aust Dent J 1997;42:11-7. 20. Maloth, S., Padamashree, S., Rema, J., Yalsangi, S., Ramadoss, T., and Kalladka, M. Diagnosis of Crouzon's syndrome. Hong Kong Dent J 2010;7:95-100. 21. Padmanabhan, V., Hegde, A.M., and Rai, K. Crouzon's syndrome: A review of literature and case report. Contemp Clin Dent 2011;2:211-4.

22. Carinci, F., Pezzetti, F., Locci, P., Becchetti, E., Carls, F., Avantaggiato, A., Becchetti, A., Carinci, P., Baroni, T., and Bodo, M. Apert and Crouzon syndromes: clinical findings, genes and extracellular matrix. J Craniofac Surg 2005;16:361-8. 23. Golabi, M., Edwards, M.S., and Ousterhout, D.K. Craniosynostosis and hydrocephalus. Neurosurgery 1987;21:63-7. 24. Lupski, J.R., Reid, J.G., Gonzaga-Jauregui, C., Rio Deiros, D., Chen, D.C., Nazareth, L., Bainbridge, M., Dinh, H., Jing, C., Wheeler, D.A., McGuire, A.L., Zhang, F., Stankiewicz, P., Halperin, J.J., Yang, C., Gehman, C., Guo, D., Irikat, R.K., Tom, W., Fantin, N.J., Muzny, D.M., and Gibbs, R.A. Wholegenome sequencing in a patient with Charcot-Marie-Tooth neuropathy. N Engl J Med 2010;362:1181-91. 25. Gilissen, C., Hoischen, A., Brunner, H.G., and Veltman, J.A. Unlocking Mendelian disease using exome sequencing. Genome Biology 2011;12:228. 26. Ng, S.B., Nickerson, D.A., Bamshad, M.J., and Shendure, J. Massively parallel sequencing and rare disease. Hum Mol Genet 2010;19:R119-24. 27. Baek, J.I., Oh, S.K., Kim, D.B., Choi, S.Y., Kim, U.K., Lee, K.Y., and Lee, S.H. Targeted massive parallel sequencing: the effective detection of novel causative mutations associated with hearing loss in small families. Orphanet J Rare Dis 2012;7:60. 28. Steinberger, D., Vriend, G., Mulliken, J.B., and Muller, U. The mutations in FGFR2-associated craniosynostoses are clustered in five structural elements of immunoglobulin-like domain III of the receptor. Hum Genet 1998;102:145-50. 29. Johnson, D. and Wilkie, A.O. Craniosynostosis. Eur J Hum Genet 2011;19:369-76. 30. Jabs, E.W., Li, X., Scott, A.F., Meyers, G., Chen, W., Eccles, M., Mao, J.I., Charnas, L.R., Jackson, C.E., and Jaye, M. Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet 1994;8:275-9. 31. Reardon, W., Winter, R.M., Rutland, P., Pulleyn, L.J., Jones, B.M., and Malcolm, S. Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet 1994;8:98-103. 32. Przylepa, K.A., Paznekas, W., Zhang, M., Golabi, M., Bias, W., Bamshad, M.J., Carey, J.C., Hall, B.D., Stevenson, R., Orlow, S., Cohen, M.M., Jr., and Jabs, E.W. Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome. Nat Genet 1996;13:492-4. 33. Meyers, G.A., Orlow, S.J., Munro, I.R., Przylepa, K.A., and Jabs, E.W. Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans. Nat Genet 1995;11:462-4. 34. Muenke, M., Gripp, K.W., McDonald-McGinn, D.M., Gaudenz, K., Whitaker, L.A., Bartlett, S.P., Markowitz, R.I., Robin, N.H., Nwokoro, N., Mulvihill, J.J., Losken, H.W., Mulliken, J.B., Guttmacher, A.E., Wilroy, R.S., Clarke, L.A., Hollway, G., Ades, L.C., Haan, E.A., Mulley, J.C., Cohen, M.M., Jr., Bellus, G.A., Francomano, C.A., Moloney, D.M., Wall, S.A., Wilkie, A.O., and Zackai, E.H. A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome. Am J Hum Genet 1997;60:555-64. 35. el Ghouzzi, V., Le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A.L., Munnich, A., and Bonaventure, J. Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat Genet 1997;15:42-6. 36. Howard, T.D., Paznekas, W.A., Green, E.D., Chiang, L.C., Ma, N., Ortiz de Luna, R.I., Garcia Delgado, C., GonzalezRamos, M., Kline, A.D., and Jabs, E.W. Mutations in TWIST,

Genetic diagnosis of craniosynostosis using WES a basic helix-loop-helix transcription factor, in SaethreChotzen syndrome. Nat Genet 1997;15:36-41. 37. Twigg, S.R., Kan, R., Babbs, C., Bochukova, E.G., Robertson, S.P., Wall, S.A., Morriss-Kay, G.M., and Wilkie, A.O. Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome. Proc Natl Acad Sci U S A 2004;101:8652-7. 38. Muenke, M., Schell, U., Hehr, A., Robin, N.H., Losken, H.W., Schinzel, A., Pulleyn, L.J., Rutland, P., Reardon, W., Malcolm, S., and Winter, R.M. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat Genet 1994;8:269-74. 39. Huang, N., Pandey, A.V., Agrawal, V., Reardon, W., Lapunzina, P.D., Mowat, D., Jabs, E.W., Van Vliet, G., Sack, J., Fluck, C.E., and Miller, W.L. Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis. Am J Hum Genet 2005;76:729-49. 40. Perlyn, C.A. and Marsh, J.L. Craniofacial dysmorphology of Carpenter syndrome: lessons from three affected siblings. Plast Reconstr Surg 2008;121:971-81. 41. Merrill, A.E., Bochukova, E.G., Brugger, S.M., Ishii, M., Pilz, D.T., Wall, S.A., Lyons, K.M., Wilkie, A.O., and Maxson, R.E., Jr. Cell mixing at a neural crest-mesoderm boundary and deficient ephrin-Eph signaling in the pathogenesis of craniosynostosis. Hum Mol Genet 2006;15:1319-28. 42. Vega, H., Waisfisz, Q., Gordillo, M., Sakai, N., Yanagihara, I., Yamada, M., van Gosliga, D., Kayserili, H., Xu, C., Ozono, K., Jabs, E.W., Inui, K., and Joenje, H. Roberts syndrome is caused by mutations in ESCO2, a human homolog of yeast ECO1 that is essential for the establishment of sister chromatid cohesion. Nat Genet 2005;37:468-70.

483

43. Vortkamp, A., Gessler, M., and Grzeschik, K.H. GLI3 zincfinger gene interrupted by translocations in Greig syndrome families. Nature 1991;352:539-40. 44. Li, L., Krantz, I.D., Deng, Y., Genin, A., Banta, A.B., Collins, C.C., Qi, M., Trask, B.J., Kuo, W.L., Cochran, J., Costa, T., Pierpont, M.E., Rand, E.B., Piccoli, D.A., Hood, L., and Spinner, N.B. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet 1997;16:243-51. 45. Carta, C., Pantaleoni, F., Bocchinfuso, G., Stella, L., Vasta, I., Sarkozy, A., Digilio, C., Palleschi, A., Pizzuti, A., Grammatico, P., Zampino, G., Dallapiccola, B., Gelb, B.D., and Tartaglia, M. Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype. Am J Hum Genet 2006;79:129-35. 46. Van Maldergem, L., Siitonen, H.A., Jalkh, N., Chouery, E., De Roy, M., Delague, V., Muenke, M., Jabs, E.W., Cai, J., Wang, L.L., Plon, S.E., Fourneau, C., Kestila, M., Gillerot, Y., Megarbane, A., and Verloes, A. Revisiting the craniosynostosis-radial ray hypoplasia association: Baller-Gerold syndrome caused by mutations in the RECQL4 gene. J Med Genet 2006;43:148-52. 47. Drera, B., Ritelli, M., Zoppi, N., Wischmeijer, A., Gnoli, M., Fattori, R., Calzavara-Pinton, P.G., Barlati, S., and Colombi, M. Loeys-Dietz syndrome type I and type II: clinical findings and novel mutations in two Italian patients. Orphanet J Rare Dis 2009;4:24. 48. Jabs, E.W., Muller, U., Li, X., Ma, L., Luo, W., Haworth, I.S., Klisak, I., Sparkes, R., Warman, M.L., Mulliken, J.B., Snead, M.L., and Maxson, R. A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 1993;75:443-50.