De Baere, Elfride

Jitendra, Jethani

Nallathambi, Jeyabalan

Neethirajan, Guruswamy

Sundaresan, Periasamy

Usha, Kim

Journal of Genetics

English

Ghent University Academic Bibliography

c© Indian Academy of SciencesRESEARCH NOTEFOXL2mutations in Indian families withblepharophimosis–ptosis–epicanthus inversus syndromeJEYABALAN NALLATHAMBI1, GURUSWAMY NEETHIRAJAN1, KIM USHA2, JETHANI JITENDRA3,ELFRIDE DE BAERE4 and PERIASAMY SUNDARESAN1∗1Department of Genetics, Dr. G.Venkataswamy Eye Research Institute, Aravind Medical Research Foundation,Aravind Eye Hospital, Madurai 625020 India2Orbit Clinic, Aravind Eye Hospital, Madurai 625020 India3Department of Pediatric Ophthalmology and Strabismus, Aravind Eye Hospital, Madurai 625020 India4Center for Medical Genetics, Ghent University Hospital, B-9000 Ghent, BelgiumIntroductionBlepharophimosis–ptosis–epicanthus inversus syndrome(BPES) (MIM#110100) is an autosomal dominant geneticcondition characterized by a complex eyelid malformation.The main features of BPES are shortening of the horizon-tal palpebral fissures (blepharophimosis), congenital ptosis,telecanthus and epicanthus inversus, and variable expressiv-ity of female infertility (premature ovarian failure) (POF) intype I BPES while absent in type II (Zlotogora et al. 1983).Both types of BPES are caused by mutations in theFOXL2 gene (MIM# 605597), consisting of a single exon of2.7 kb located on chromosome 3q23 (Crisponi et al. 2001).It encodes a protein that belongs to the family of winged-helix/forkhead transcription factors that contains 100 aminoacid DNA-binding forkhead domain and a polyalanine tract.FOXL2 expression is restricted to developing eyelids of fetaland adult ovarian granulosa cells (Cocquet et al. 2003).In previous mutation studies intragenic mutations werefound in 70% of patients (De Baere et al. 2003). Recently, insporadic and familial BPES cases five microdeletions werefound outside the coding region of FOXL2. In addition, 11rearrangements encompassing FOXL2 have been described.Overall, genomic rearrangements encompassing or outsidethe FOXL2 account for 16% of all molecular defects foundin BPES families (Beysen et al. 2005).Genotype–phenotype correlations suggested that FOXL2mutations resulting in predicted proteins with a truncation*For correspondence. E-mail: sundar@aravind.org.before the polyalanine tract might lead to development ofthe POF (BPES type I). However, correlations for mutationsleading to a truncated or extended protein containing an in-tact forkhead domain and polyalanine tract, and for missensemutations in the highly conserved forkhead domain, are stillelusive. Large submicroscopic deletions may result in BPESassociated with mental retardation (De Baere et al. 2003;Beysen et al. 2005).Here we report the identification of intragenic FOXL2mutations in two Indian families with BPES type I and II re-spectively. We demonstrate that our results are in agreementwith previously reported genotype–phenotype correlations.Materials and methodsPatients and clinical evaluationRecruitment was based on interviews, questionnaires, andclinical examination of aﬀected and unaﬀected individualsby ophthalmologists. An informed consent was obtained incompliance with the Helsinki Declaration. Clinical data areshown in table 1. The pedigrees of the two families areshown in figure 1a and figure 2a. A four-generation BPEStype I family with 14 aﬀected members of which nine arealive, and were selected for genetic analysis (figure 1a). Theproband was a one-year-old girl with typical BPES and notcooperative for visual acuity testing. However, adapted vi-sual acuity testing revealed an acuity of atleast 6/24 or better.Cycloplegic refraction revealed +0.5 D for the right eye and+1.0 D for the left eye.Keywords. BPES syndrome; FOXL2 gene; haploinsuﬃciency; premature ovarian failure (POF); FOXL2 polyalanine expansion; geneticcounseling.Journal of Genetics, Vol. 86, No. 2, August 2007 165Jeyabalan Nallathambi et al.Figure 1. a. Four-generation BPES type I pedigree. All aﬀected members have typical BPES. Threeaﬀected females from generations I and II are infertile. Aﬀected individual BPES III:1 has mental re-tardation and microcephaly. The aﬀected and unaﬀected patients analysed molecularly are indicatedby numbers “IV:1, III:3, etc”. An asterisk indicates the two aﬀected individuals having mental retar-dation and microcephaly. b. Sequence electropherogram showing the heterozygous FOXL2 mutationc.273C > G (p.Y91X) found in aﬀected members of this BPES type I family. c. Segregation analysis inaﬀected and unaﬀected family members using BseLI restriction enzyme digestion. The BseLI restric-tion enzyme showed 175 bp and 113 bp in the wild type allele while additional fragments of 82 bp and93 bp were observed in mutant alleles. These fragments were separated on a 2.5% agarose gel.Another three-generation BPES type II family had threeaﬀected members, with a three-year-old male proband with atypical BPES (figure 2a). Visual acuity recorded by Cardiﬀacuity chart and Teller preferential looking charts was 6/9 inboth eyes. The cycloplegic refraction was +1.0 D in botheyes. The probands of both the families underwent frontalissling surgery in both the eyes for ptosis and Y-V plasty fortelecanthus correction.Mutation analysisBlood samples were collected from consenting individualsfollowed by extraction of genomic DNA following a stan-dard protocol. The coding region of the FOXL2 gene wasPCR amplified using previously reported primers (De Baereet al. 2003; Kumar et al. 2004). The PCR amplicons weresequenced bidirectionally (3730 ABIPRISM Analyzer, BigDye Terminator, Applied Biosystems) followed by RFLPanalysis. The segregation of the mutation in the BPES typeI family was assessed by restriction enzyme digestion us-ing 2 units of BseLI (MBI FERMANTAS) followed by elec-trophoresis on a 2.5% agarose gel (figure 1c).Results and discussionIn a four-generation BPES type I family with nine living af-fected members, three aﬀected females are infertile and twoaﬀected male patients (BPES III:1 and his younger brother)have mental retardation with microcephaly (figure 1a). Inall the aﬀected individuals analysed a nonsense mutationc.273C > G (p.Y91X) was identified in FOXL2, leading toa predicted truncated protein of 90 amino acids (instead of376) with a partial forkhead domain and polyalanine tract isabsent (figure 1b,c).These mutations are supposed to lead to haploinsuﬃ-ciency of FOXL2, since DNA-binding activity is abolisheddue to disruption of the forkhead domain. Although ingeneral, transcripts with nonsense mutation are degradedby nonsense-mediated decay (NMD), intronless genes asthe FOXL2 gene might escape NMD, giving rise to astable mutated transcripts and expressed mutated proteins(Frischmeyer and Dietz 1999).In general, mutations leading to the predicted proteinswith a partial forkhead domain are expected to lead to BPEStype I as suggested previously (De Baere et al. 2003). Indeedthis nonsense mutation leads to BPES type I in this family.166 Journal of Genetics, Vol. 86, No. 2, August 2007F0XL2 gene mutationsTable1.ClinicalfeaturesofIndianfamilieswithblepharophimosis-ptosis-epicanthusinversussyndrome.BPESAﬀectedAge/SexBPESICDOCDPFL(mm)MentalMicrocephalyInfertilityConsanguinityfamiliesindividuals(mm)(mm)RELEretardationFourIV:21/F+33743.54--Unknown-generationIII:232/M+358833.5---+Type1III:527/M+NMNMNMNM---+(figure1)I:190/F+369246.5--+-II:168/M+NMNMNMNM----II:265/F+NMNMNMNM--+-II:462/M+389022----III:140/M+NMNMNMNM++-+III:610/M+NMNMNMNM----ThreeII:153/M+33905.55.5----generationIII:127/F+30946.56.5----TypeIIIV:13/M+337867----(figure2)ICD,innercanthaldistance;OCD,outercanthaldistance;PFL,palpebralfissurelength;BE,righteye,LE,lefteye;NM;notmeasured“+”,Present;“-”:absent.Figure 2. a. Three-generation BPES type II pedigree. The af-fected and unaﬀected patients analysed at the molecular level areindicated by numbers “II:1, III:1, etc”. b. Segregation analysis ofthe 30-bp FOXL2 duplication c.672 701dup “g.909 938” using gelelectrophoresis on a 2.5% agarose gel, in aﬀected (heterozygousPCR fragment of 304 bp and 334 bp) and unaﬀected family mem-bers (homozygous fragment of 304 bp).Interestingly, two aﬀected brothers have BPES with mentalretardation and microcephaly (figure 1a and table 1). Theyounger brother died and could not be clinically examinedto undergo molecular testing; the older one however (BPESIII:1) was shown to carry the familial FOXL2 mutation. Itis not clear whether these associated features are caused bythe familial FOXL2 mutation in these brothers. IntragenicFOXL2 mutations are known to occur in BPES individualswith normal psychomotor development and normal head cir-cumference (De Baere et al. 2003). It is important to men-tion that these mentally retarded brothers descend from aconsanguineous marriage, their parents being first cousins.Thus it cannot be excluded that the mental retardation andmicrocephaly result from a diﬀerent recessive condition in-dependent from BPES. Notably, one of the aﬀected females(proband BPES IV:2) is a one-year-old female of whom theBPES type cannot be assessed due to her prepubertal age (ta-ble 1). However, she is predicted to develop BPES type I,which has implications for genetic counseling.In addition we identified a recurrent 30-bp duplicationJournal of Genetics, Vol. 86, No. 2, August 2007 167Jeyabalan Nallathambi et al.c.672 701dup “g.909 938” in the FOXL2 gene in a three-generation family aﬀected by BPES type II (figure 2a). Thisduplication cosegregated with the disease in this family (fig-ure 2b). This mutation leads to an inframe polyalanine ex-pansion in the FOXL2 protein (p.A224 A234 dup). It hasbeen reported as a pathogenic change in sporadic and fa-milial cases in other ethnicities, but this is the first reportin an Indian pedigree with BPES (Human FOXL2 MutationDatabase; http://mebgen.ugenp.be/foxl2).This polyalanine expansion represents more than 30% ofintragenic FOXL2 mutations. It is predicted to result in a hy-pomorphic allele and lead to BPES type II. Recently, it wasproposed that the pathogenic mechanism of this polyalanineexpansion may be its mislocalisation (due to cytoplasmic ag-gregation) together with its inclusion into nuclear aggregates(Caburet et al. 2004). We have recently identified a shorthomozygous polyalaline expansion (of 19 alalines instead of14) in a consanguineous Indian pedigree, resulting in a reces-sive form of BPES and ovarian dysfunction (Nallathambi etal. 2007). The findings in our family lend further support tothe genotype–phenotype correlation for this type of mutation(polyalanine expansion).In summary, we have reported a nonsense mutation ina BPES type I family and a recurrent duplication leadingto a polyalanine expansion in a BPES type II family, bothare of Indian origin. This is the first study where genotype–phenotype correlations have been possible in Indian BPESfamilies. They are in agreement with previous mutation stud-ies in BPES. Genotype–phenotype correlations in BPES areespecially useful for predictive testing of POF risk in aﬀectedfemales.AcknowledgementsThe authors are grateful to the participants of this study. Authorsthank Dr V. R. Muthukkaruppan, Director of Research, AravindMedical Research Foundation for his valuable suggestions. We alsothank V. R. Muthulakshmi and B. Suganthi for sample collection,T. P. Vasanthi, for technical assistance and Mr R. Jeyakrishnan &Mr. M. Rajkumar for photographic work.ReferencesBeysen D., Raes J., Leroy B. P., Lucassen A., Yates J. R., Clayton-Smith J., Ilyina H. et al. 2005 Deletions Involving Long-RangeConserved Nongenic Sequences Upstream and Downstream ofFOXL2 as a Novel Disease-Causing Mechanism in Blepharophi-mosis Syndrome. Am. J. Hum. Genet. 77, 205–218.Caburet S., Demarez A., Moumne L., Fellous M., De Baere E. andVeitia R. A. 2004 A recurrent polyalanine expansion in the tran-scription factor FOXL2 induces extensive nuclear and cytoplas-mic protein aggregation. J. Med. Genet. 41, 932–936.Cocquet J., De Baere E., Gareil M., Pannetier M., Xia X., FellousM. and Veitia R. A. 2003 Structure, evolution and expressionof the FOXL2 transcription unit. Cytogenet. Genome Res. 101,206–211.Crisponi L., Deiana M., Loi A., Chiappe F., Uda M., Amati P., Bis-ceglia L. et al. 2001 The putative forkhead transcription factorFOXL2 is mutated in blepharophimosis/ptosis/epicanthus inver-sus syndrome. Nat. Genet. 27, 159–166.De Baere E., Beysen D., Oley C., Lorenz B., Cocquet J., De Sut-ter P., Devriendt K. et al. 2003 FOXL2 and BPES: mutationalhotspots, phenotypic variability, and revision of the genotype–phenotype correlation. Am. J. Hum. Genet. 72, 478–487.Frischmeyer P. A. and Dietz H. C 1999 Nonsense-mediated mRNAdecay in health and disease. Hum. Mol. Genet. 8, 1893–1900.Kumar A., Babu M., Raghunath A. and Venkatesh C. P. 2004 Ge-netic analysis of a five generation Indian family with BPES: anovel missense mutation (p.Y215C). Mol. Vis. 10, 445–449.Nallathambi J., Moumne L., De Baere E., Beysen D., Usha K., Sun-daresan P. and Veitia R. A. 2007 A novel polyalanine expansionin FOXL2: the first evidence for a recessive form of the ble-pharophimosis syndrome (BPES) associated with ovarian dys-function. Hum. Genet. 121, 107–112.Zlotogora J., Sagi M. and Cohen T. 1983 The blepharophimosis,ptosis, and epicanthus inversus syndrome: delineation of twotypes. Am. J. Hum. Genet. 35, 1020–1027.Received 27 January 2006168 Journal of Genetics, Vol. 86, No. 2, August 2007

FOXL2 mutations in Indian families with blepharophimosis-ptosis-epicanthus inversus syndrome

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FOXL2 mutations in Indian families with blepharophimosis-ptosis-epicanthus inversus syndrome

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