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Short Communication
Expanding the mutation spectrum for Fraser syndrome: Identificationof a novel heterozygous deletion in FRAS1
Julia Hoefele a,⁎, Christian Wilhelm b, Monika Schiesser c, Reinhold Mack d, Uwe Heinrich a, Lutz T. Weber e,Saskia Biskup b, Cornelia Daumer-Haas f, Hanns-Georg Klein a, Imma Rost a
a Center for Human Genetics and Laboratory Medicine, Martinsried, Germanyb CeGaT GmbH, Tübingen, Germanyc Medical Practice in Prenatal Medicine, Munich, Germanyd Gynecology Practice, Ingolstadt, Germanye Pediatric Nephrology, Dr. von Haunersches Kinderspital, University Children's Hospital, Ludwig Maximilians University Munich, Germanyf Prenatal Medicine Munich, Munich, Germany
Abbreviations: FS, Fraser syndrome; ECM, extracellulparaffin embedded; Array-CGH, array-based comparativecongenital anomalies of the kidney and urinary tract; MLPprobe amplification.⁎ Corresponding author at: Center for Human Gene
Lochhamer Str. 29, 82152 Martinsried, Germany. Tel.: +895578 780.
Fraser syndrome (FS) is a rare autosomal recessive inherited disorder characterized by cryptophthalmos, la-ryngeal defects and oral clefting, mental retardation, syndactyly, and urogenital defects. To date, 250 patientshave been described in the literature. Mutations in the FRAS1 gene on chromosome 4 have been identified inpatients with Fraser syndrome. So far, 26 mutations have been identified, most of them are truncating muta-tions. The mutational spectrum includes nucleotide substitutions, splicing defects, a large insertion, and smalldeletions/insertions. Moreover, single heterozygous missense mutations in FRAS1 seem to be responsible fornon-syndromic unilateral renal agenesis.Here we report the first case of a family with two patients affected by Fraser syndrome due to a deletion of64 kb (deletion 4q21.21) and an additional novel frameshift mutation in exon 66 of the FRAS1 gene. Todate, large deletions of the FRAS1 gene have not yet been described. Large deletions seem to be a rare causefor Fraser syndrome, but should be considered in patients with a single heterozygous mutation.
Fraser syndrome (FS; OMIM#219000) is a rare autosomal recessivedisorder characterized by cryptophthalmos, syndactyly, abnormalitiesof the respiratory and urogenital tract (Fraser, 1962; Slavotinek andTifft, 2002; van Haelst et al., 2007). Thomas et al. implemented diag-nostic criteria for FS dividing into major criteria (cryptophthalmos;syndactyly; abnormal genitalia; an affected sibling) and minor criteria(congenital malformation of the nose, ears and larynx; cleft lip/palate;skeletal malformations; renal agenesis; mental retardation; umbilicalhernia) (Thomas et al., 1986; vanHaelst et al., 2007). FS can be diagnosed,if two major and one minor criteria or one major and four minor criteriaare present (van Haelst et al., 2007). FS shows an interfamilial highly var-iable phenotype ranging from minor symptoms to lethal malformationslike renal agenesis. On the other hand, a strong phenotypic similarity
ar matrix; FFPE, formalin fixedgenomic hybridization; CAKUT,A, multiplex ligation-dependent
tics and Laboratory Medicine,49 89 895578 0; fax: +49 89
de (J. Hoefele).
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exists within a family (Slavotinek and Tifft, 2002). The incidence of FShas been found to be 0.43 per 100,000 live births (Martinez-Frias et al.,1994). Mutations in the FRAS1, FREM2, and GRIP1 gene have been identi-fied causing FS (McGregor et al., 2003; Vogel et al., 2012). Mutations inFRAS1 are responsible for the classical phenotype of FS (McGregor et al.,2003). FREM1mutations can also be associated with a less severe pheno-type as seen in Manitoba Oculotrichoanal Syndrome (MOTA; OMIM#248450) and bifid nose, with or without anorectal and renal anomalies(BNAR; OMIM #608980) (Vogel et al., 2012). Mutations in FREM2 canalso be found in a subset of FS patients suggesting heterogeneity of thissyndrome (Smyth and Scambler, 2005). Almost 50% of the patientsexhibit mutations in either of the genes. Themolecular defect underlyingthe other half still remains unknown. The FRAS1 gene is located on chro-mosome 4q21.21 and encodes a protein that is widely expressed. It hassequence similarity to genes encoding for a set of extracellular matrix(ECM) proteins (McGregor et al., 2003). Up to now, nucleotide substitu-tions, splicing defects, a large insertion, and small deletions/insertionshave been described to be responsible for FS. 26 mutations are listedin HGMD (http://www.biobase-international.com), most of them aretruncating.
Here we report the first case of a family with two fetuses affectedby FS carrying – besides a novel frameshift mutation – a deletion ofseveral exons of the FRAS1 gene. To our knowledge, large deletions
Fig. 2. Fragments of arms and legs of the second fetus showing syndactyly between thefingers of the right hand and the toes of both feet (with kind permission of Dr. I.Kleinlein, Institute of Pathology,Municipal HospitalMunich-Harlaching,Munich, Germany).
195J. Hoefele et al. / Gene 520 (2013) 194–197
in FRAS1 have not yet been described. Deletions of the FRAS1 genetherefore seem to be a rare cause for FS.
2. Patient and methods
2.1. Case report
The family is of German origin and presented to our department in2012 for genetic counseling (pedigree is shown in Fig. 1). The medicalhistory revealed a tubal pregnancy in 1995. During the second preg-nancy in 2005 prenatal ultrasound showed a female fetus with renalagenesis on the left side and a multicystic dysplastic kidney on theright side. The thorax seemed hypoplastic and there was anhydramnia.The fetus was stillborn at 29 weeks of gestation (Fig. 1). Post morteminvestigations additionally showed bilateral anophthalmia, bilateralcleft lip and palate, tracheal stenosis, and an anal atresia type II. Unfor-tunately, pictures from this fetus do not exist. Retrospectively, wemadethe clinical diagnosis of a FS in 2012 and initiated genetic analysis of thefetus and the parents. While molecular examination of the fetus wasongoing, the mother became pregnant again. Ultrasonography at a ges-tational age of eleven weeks showed a female fetus with bilateral cleftlip and palate. Post mortem investigations at a gestational age of thir-teen weeks (induced abortion) showed a syndactyly of the second tothe fourth finger on the right hand and between the second and thefifth toe on both feet (see Fig. 2). Additionally, an atresia of the epiglottisresulting in an occlusion of the trachea could be found. Further mal-formations have not been seen because of extended fragmentationof the fetal tissue. The further medical history of the family was un-remarkable. Both parents did not show any renal or urinary tract mal-formation in the ultrasonography examination.
2.2. Mutational analysis
Tissues of both fetuses and blood samples of the parents were col-lected after written informed consent. Genomic DNA of the first fetuswas extracted from formalin fixed paraffin embedded (FFPE) tissueusing the NucleoSpin FFPE DNA kit (Macherey Nagel), genomicDNA of the second fetus was extracted from chorionic villi usingthe NucleoSpin Blood DNA kit (Macherey Nagel, Dueren, Germany).Genomic DNA of the parents was extracted from blood using theGentra Puregene Blood Kit (Qiagen, Hilden, Germany). Because ofpoorDNAquality of thefirst fetus, direct sequencingwasfirst performedin the parents for all 74 FRAS1 exons on both strands using the dideoxychain termination method on an ABI capillary sequencer 3730 (AppliedBiosystems, Foster City, USA). Primers were designed by Primer3program (http://frodo.wi.mit.edu/primer3/input.htm). DNA alignmentand sequence variant analysis were carried out using the SequencePilotCE software (JSI Medical Systems GmbH, Kippenheim, Germany).The DNA sequence analysis of both fetuses was done by targeted diag-nostic sequencing.
Fig. 1. Pedigree of the family. Circle female, square male, arrow affected fetuses. Bothparents are heterozygous for a FRAS1 mutation.
2.3. Array-CGH
Oligo array-CGH (array-based comparative genomic hybridization)was performed in the mother and both fetuses using the CytoChipISCA 4 × 180 k oligonucleotide microarray containing about 180,00045-60-mer oligo and control probes (BlueGnome, Cambridge, UK)with an average resolution of 25 kb in targeted and 100 kb in backboneregions. After the fluorescent labeling the Cy3-labeled test DNA and aCy5-labeled sex-mismatched reference DNA (Promega, Mannheim)were hybridized on the array according to the manufacturer's protocol.The array was analyzed using the BlueFuse Multi software version 2.6(BlueGnome, Cambridge, UK) based on the Human Genome Assembly,February 2009 (hg19), Ensembl Release 68.
3. Results
Mutational analysis of the first fetus could not be performed for allexons of the FRAS1 gene because of poor DNA quality. Therefore, mu-tational analysis was first done in the parents. In exon 66 of the FRAS1gene the novel heterozygous mutation c.10346delA resulting in a pre-mature stop (p.Glu3449GlyfsX2) was identified in the father (Fig. 3).Targeted diagnostic sequencing revealed that the first fetus was carrierof the paternal mutation. Examination of the second fetus, which wasperformed during pregnancy at thirteenth weeks of gestation, showedthe paternal mutation as well (Fig. 3). The mother did not show anycausative mutation in the FRAS1 gene, but presented with multiplehomozygous polymorphisms, which were located between exons 6–9and 25–71. Heterozygous polymorphisms were only detected in exons1, 2, and 17.
Because of the unexpected accumulation of homozygous polymor-phisms in the mother, array-CGH was additionally performed and re-vealed a heterozygous 63.87 kb interstitial deletion on chromosome4q21.21 (Fig. 4). The minimal deleted region extends from 79,125,989to 79,189,854 and contains the exons 3 to 9 of the FRAS1 gene. Themaximum deleted region extends from rs79,108,116 to 79,209,252 in-cluding the exons 3–14 (Fig. 4). Array-CGH of both fetuses showedthe deletion of the FRAS1 gene as well (Fig. 5).
4. Discussion
Fraser syndrome is characterized by cryptophthalmus, syndactyly,and urogenital defects (Slavotinek et al., 2006). So far, all knownFRAS1mutations are missense or nonsense mutations, splicing defect,small deletions and insertions. FRAS1 mutations can be identified in40–50% of the patients. Slavotinek first described a patient with asinglemutation in the FRAS1 gene (Slavotinek et al., 2006). A furthermo-lecular study of 33 families with FS identified two non-consanguineous
Fig. 3. Partial nucleotide sequence of exon 66 of the FRAS1 gene from the second fetus,the father, and a healthy control. Fetus and father are both heterozygous for the muta-tion c.10346delA resulting in p.Glu3449GlyfsX2.
Fig. 4. Array-CGH chromosome detailed view profile of chromosome 4 comprising thedeletion on 4q21.21 in the mother. On the left, the partial chromosome 4 ideogram.
Fig. 5. Array-CGH chromosome detailed view profile of chromosome 4 comprising thedeletion on 4q21.21 in the second fetus. On the left, the partial chromosome 4 ideogram.
196 J. Hoefele et al. / Gene 520 (2013) 194–197
families with a singlemutation in FRAS1 (van Haelst et al., 2008). Severalexplanations for these findings have then been postulated: i) one muta-tion has been missed by Sanger sequencing; ii) coding exons, whichmight be unknown, were not included in the analysis; iii) a secondmutation in another gene is missing. The latter would be consistentwith a digenic mode of inheritance. A similar finding of two heterozy-gous mutations in two different genes has been reported in some renaldiseases, i.e. nephronophthisis or Bardet–Biedl syndrome, but has notbeen reported for FS (Hoefele et al., 2007; Katsanis et al., 2001). A possi-ble explanation for the missing second mutation would be our observa-tion of a heterozygous deletion in the FRAS1 gene, which cannot be seenby Sanger sequencing.
Saisawat et al. observed single heterozygous missense mutations ofFRAS1 in three patients with non-syndromic unilateral renal agenesis.All identified mutations were absent from 96 healthy control individ-uals and were classified as possible damaging using the mutation pre-diction program PolyPhen-2. Because there is evidence of phenotypicexpression in heterozygotes of some recessive diseases, they postulatedthat heterozygous mutations in FRAS1 cause non-syndromic CAKUT(congenital anomalies of the kidney and urinary tract) (Saisawat et al.,2012). A deletion or duplication analysis using array-CGH was notperformed in these patients. As both parents in our case report –
although heterozygous – did not show any renal or urinary tractmalfor-mation, it seems likely that heterozygosity does not lead to a renal phe-notype. One possible hypothesis could be that the patients described in
Saisawat et al. might have an additional causative mutation, which wasmissed by Sanger sequencing.
The FS phenotype of our patients presented here appears to be in-distinguishable from the phenotype observed in patients with alreadyknown types of mutation. Patients with a single mutation in FRAS1should be examined by array-CGH as of now. A commercial MLPAprobe set for the detection of large deletions or duplications in FRAS1is not available so far.
In conclusion, this report describes the first large deletion as causefor FS in humans in addition to a novel frameshift mutation. Both par-ents are carrier for one of the mutations. Our findings expand thespectrum of causative mutations in FS and the possibility for diagnos-tic testing and prenatal diagnosis for FS patients and their families.
Acknowledgments
We would like to thank the family for participation.
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