Microdeletions at Chromosome Bands 1q32-q41 as aCause of Van der Woude Syndrome

Brian C. Schutte,1 Ann M. Basart,1 Yoriko Watanabe,1 Jennifer J.S. Laffin,2 Kevin Coppage,1Bryan C. Bjork,2 Sandy Daack-Hirsch,1 Shiva Patil,1 Michael J. Dixon,3 and Jeffrey C. Murray1,2,4*1Department of Pediatrics, University of Iowa, Iowa City, Iowa2Program in Genetics, University of Iowa, Iowa City, Iowa3School of Biological Sciences and Departments of Dental Medicine and Surgery, University of Manchester,Manchester, United Kingdom

4Department of Biology, University of Iowa, Iowa City, Iowa

Van der Woude syndrome (VWS) is an auto-somal dominant disorder comprising cleftlip and/or cleft palate and lip pits. We re-ported previously a family whose underly-ing mutation is a 500–800 kb deletion local-ized to chromosome bands 1q32-q41 [Sanderet al., 1994: Hum Mol Genet 3:576–578]. Alongwith cleft lip/palate and lip pits, affectedrelatives exhibit developmental delays, sug-gesting that the function of a gene nearbymay also be disrupted. To further localizethe VWS gene we searched for other dele-tions that cause VWS. An allele loss assaywas performed using a novel highly poly-morphic marker, D1S3753. From a panel of37 unrelated individuals, we detected an al-lele loss in one family, indicating the pres-ence of a deletion. In this family, the pheno-type in three generations of affected indi-viduals was confined to the cardinal signs ofVWS. Surprisingly, mapping of the new de-letion showed that it extended 0.2–1 Mb be-yond the proximal breakpoint for the dele-tion described previously. No deletionswere detected in seven cases of poplitealpterygia syndrome, 76 cases of mixed syn-dromic forms of cleft lip and palate, and 178cases of nonsyndromic cleft lip and palate.These observations suggest that geneticsearches for microdeletions should be rou-tine in screening patients for causes of VWSand may facilitate the positional cloning ef-forts of the VWS gene and of a nearby gene

or genes that may be involved in brain de-velopment. Am. J. Med. Genet. 84:145–150,1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: Van der Woude syndrome;microdeletion; 1q32; cleft lip;cleft palate; lip pits; develop-mental delay

INTRODUCTION

Van der Woude syndrome (VWS) is an autosomaldominant disorder characterized by cleft lip and/or pal-ate and lip pits with occasional hypodontia (OMIM no.119300). The disorder was first described in the nine-teenth century and was characterized in detail by AnnVan der Woude [1954]. Bocian and Walker [1987] re-ported a large interstitial deletion at chromosomebands 1q32-q41 in a child with VWS, and additionalanomalies. Linkage of VWS to chromosome 1 was firstsuggested by Wienker et al. [1987] to the Duffy bloodgroup and was subsequently confirmed by Murray etal. [1990]. Sander et al. [1994] reported a microdeletionin a single family with VWS in which affected individu-als exhibited developmental delay along with the typi-cal phenotype of VWS. This microdeletion confirmedand narrowed the critical region for the VWS gene andsuggested that VWS was caused by haploinsufficiencyof a gene at 1q32-q41. Thus, deletions at 1q32-q41 maybe found in other VWS families.

The presence of microdeletions in human chromo-somes as the underlying cause for Mendelian disorderswas first reported for the a and b globin loci [Otto-lenghi et al., 1974, 1976; Taylor et al., 1974; Ramirez etal., 1976] and later for Duchenne muscular dystrophy[Francke et al., 1985]. Other examples include a groupof disorders where multiple genes or sex-specific regu-latory elements are deleted to generate a recognizablephenotype such as Williams syndrome (OMIM no.194050), Angelman and Prader-Willi syndromes(OMIM no. 105830), Smith-Magenis syndrome (SMS;

Contract grant sponsor: National Institutes of Health; Contractgrant numbers: P50-DE09170, R01-DE08559, and P30-HD27748;Contract grant sponsor: Action Research; Contract grant number:S/P/3261.

*Correspondence to: Jeffrey C. Murray, Department of Pediat-rics, University of Iowa, Iowa City, IA 52242. E-mail: jeff-murray@uiowa.edu

Received 28 October 1998; Accepted 11 January 1999

American Journal of Medical Genetics 84:145–150 (1999)

© 1999 Wiley-Liss, Inc.

OMIM no. 182290) and VCF/DiGeorge syndrome(OMIM no. 188400).

Deletions that cause human disorders may be char-acterized on the basis of deletion frequency, location ofbreakpoints, and range of phenotypes caused by thedeletion. The deletion frequency is here defined as thenumber of cases of a specific syndrome whose molecu-lar etiology is a deletion divided by the total number ofcases. This value ranges from a few percent, observedat the b-globin locus [Kazazian and Boehm, 1988], togreater than 90%, observed at the Williams syndromelocus [Nickerson et al., 1995]. For loci that are fre-quently deleted, the deletion breakpoints are oftenfound to be highly clustered, suggesting a commonmechanism for their origin. An important mechanismfor their origin has been shown to be homologous re-combination between duplicated regions [Nicholls etal., 1987; Dutly and Schinzel, 1996; Urban et al., 1996;Campbell et al., 1997; Carrozzo et al., 1997; Chen et al.,1997]. Deletions cause a range of phenotypes that maybe indistinguishable from a recognizable disorder orthey may cause additional phenotypic anomalies. Asseen at the lissencephaly 1 locus (LIS1; OMIM no.601545), large deletions cause Miller-Dieker syndrome,which includes a characteristic facial phenotype in ad-dition to lissencephaly. The additional phenotypes arepresumably caused by the disruption of function of ad-jacent genes [Chong et al., 1997]. Thus, by screeningindividuals whose phenotype is restricted to the char-acteristics of the disorder, it is more likely to identifysmaller deletions that may help localize the diseasegene.

In this paper, a search for microdeletions was per-formed on a large panel of VWS families to determinetheir contribution as the molecular cause of VWS. Us-ing a single, highly polymorphic marker, one new de-letion was detected. The breakpoints of the new dele-tion were mapped to see if it would further localize theVWS gene. They were also compared to the previouslyreported microdeletion to test for the potential cluster-ing of breakpoints. The location of the breakpoints wascompared with the phenotypes caused by the deletionsto determine whether additional phenotypic changesobserved in the previous microdeletion case were due tothe deletion of a nearby gene. Finally, no deletionswere found in 261 total cases of other clefts which in-cluded popliteal pterygia (seven cases), a mixture ofother syndromic forms (76 cases) and nonsyndromicforms (178 cases), suggesting large deletions at theVWS locus are not a common mechanism for these dis-orders.

METHODSClinical Material

VWS and popliteal pterygia syndrome popula-tions. Thirty-seven VWS and seven popliteal pte-rygia syndrome (PPS) families (Table I) were ascer-tained through contact with geneticists, orthodontists,or cleft-palate clinics. All patients were examined byone or more geneticists or clinical collaborators (J.C.M.,S.D.H., Achim Sander, Ed Lammer, Denize Tiziane,Mahin Golabi, George Hoganson, Bea Fowlow, Rena

Falk, Elaine Zackai, Lewis Holmes, Michael Aldred,John Mulliken, Franklin Desposito, Dawn Delozier-Blanchet, Virginia Sybert, Holly Ardinger, R.J.M.Gardner, Rich Pauli, Victor McKusick, Albert Schinzel,Aravinda Chakravarti, Tara C. Matise, Susan Kirkpa-trick, Mark Greenstein, Louis Bartoshesky, HowardSaal, Cheryl Reid, Arthur Alysworth, Dick Lindhout,Nancy Cangany). Individuals were considered to be af-fected with VWS if they had one or more of cleft lip,cleft palate, hypodontia, or lower-lip pits and consid-ered to have PPS if they had pterygia along with any ofthe VWS findings. All of the families had multiple af-fected individuals, except family VWS26 in which theindex case had cleft lip and palate and lip pits and theparents were unaffected. Development and mental per-formance appeared to be normal in all affected indi-viduals in this study. In particular, family VWS771 hasbeen repeatedly examined (S.D.H., J.C.M.) and has noevidence of cognitive delay or physical anomalies otherthan congenital cleft lip and palate and lip pits. Ten-milliliter samples of whole blood were obtained from theadults and 1 ml whole blood/kg body weight was obtainedfrom each of the children in the families studied.

Other populations with clefts. Our study also in-cluded a panel of samples from individuals with non-syndromic clefts and a panel of samples from individu-als with syndromic clefts. Clefts were classified as non-syndromic or syndromic as described previously[Munger et al., 1996]. The nonsyndromic cleft panelwas described previously [Lidral et al., 1998], and con-tains 178 nuclear family triads from Iowa. The syn-dromic cleft panel contains 80 nuclear triad familiesfrom Iowa whose index case exhibits a syndromic cleft.This panel included a few cases of each of the followingsyndromes or chromosomal abnormalities: Stickler, ho-loprosencephaly, Pierre Robin, Goldenhar, CHARGE,EEC, Pena-Shokeir, and Hey Wells. For this study,only the syndromic cleft cases that were caused by achromosome abnormality (four total) were excludedduring data analysis, since the cause of those clefts wasknown. The syndromic cleft panel was ascertainedthrough the Iowa Birth Defects Registry and sampleswere obtained under the auspices of the University ofIowa Craniofacial Anomalies Research Center as pre-viously described for the nonsyndromic cleft panel [Li-dral et al., 1998].

Polymerase Chain Reaction

DNA was purified from whole blood using a pub-lished procedure [Miller et al., 1988]. Approximately 40ng of template DNA was analyzed by polymerase chain

TABLE I. Populations With Clefts Used to Screen forMicrodeletions at 1q32-q41

Population

Number ofsamples from

unrelatedindividuals Reference

VWS 37 This studyPPS 7 This studyNonsyndromic cleft panel 178 Lidral et al., 1998Syndromic cleft panel 76 (80 − 4) This study

146 Schutte et al.

reaction (PCR) using standard conditions (GDB no.9798291). The primer sequences for all markers usedin this study are available from GDB (Table II; www.gdb.org). These include three novel markers: D1S3753(forward, 58-CATGTAATTCAGGTTAGCATGA-38; re-verse 58-GAGCGATGATAGTGCCACTAC-38),D1S3754(forward, 58-TTCCATACTTGACCCCCTACC-38; reverse58-TTCATTGATGGTGCTTTTATGC-38), and yAS9R(forward 58-ACGGAAATCTTTAGTAAAAGGCG-38; re-verse 58-AGTAATTCTTTTTGTGCGACCTG-38), whichwill be described elsewhere (B. Bjork, B. Schutte, K.Coppage and J. Murray, manuscript in preparation).To detect sequence length polymorphisms, the ampli-fied products were separated on 6% acrylamide dena-turing gels for 2 hr at 60 W. For SSCP analysis, theamplified products were separated on MDE (FMC Bio-products) nondenaturing gels for 5–6 hr at 20 W atroom temperature with fans or for 16 hr at 6 W at 4°C.DNA bands were detected on all gels by silver staining.

Genetic Analysis

For the allele-loss assay, DNA samples were geno-typed with the novel short tandem repeat D1S3753(Table II). If individuals were mono-allelic forD1S3753, then additional relatives were analyzed tolook for loss of transmission of alleles in affectedmembers, indicative of a microdeletion. To control forsampling or processing error, nonpaternity, and isodi-somy, the family members were genotyped with sevenrandomly selected genetic markers from outside theVWS critical region. Four markers were derived fromchromosome 1, GATA165G08, D1S3728, D1S3732, andD1S3734, and three markers were derived from otherchromosomes, D2S443, D17S1290, and GATA126F09(Chr 5). To map the microdeletions, relatives weregenotyped with additional polymorphic markers fromthe VWS region (Table II).

Cytogenetic Analysis

The family that displayed an allele loss in affectedindividuals was screened for cytogenetic abnormalities.Giemsa staining on blood sample chromosomes wasperformed using standard techniques. Cells were syn-chronized at metaphase, spread on a slide, and incu-bated at 90°C for 1 hr. The slides were then trypsin-ized, stained with Wright Giemsa (GTW), and visual-ized under a microscope.

RESULTSScreen for 1q32-q41 Microdeletions

Previously, Sander et al. [1994] identified a deletionat 1q32-q41 in a VWS family that cosegregated withthe disease. This observation confirmed that VWS was

caused by haploinsufficiency, and suggested that VWSin other families may be caused by a deletion at 1q32-q41. To test this hypothesis, we screened a panel ofVWS families for loss of transmission of an allele withthe novel highly polymorphic marker D1S3753. Thismarker is a complex microsatellite repeat that contains14 copies of a TTCC repeat followed by 15 copies of a CTrepeat. It was identified by sequence analysis of a ge-nomic clone from the VWS critical region. Genotypeanalysis of a panel of 318 unrelated normal individualswith D1S3753 yielded 13 different alleles and an over-all heterozygosity of 0.88. The high heterozygosity ofthis marker suggested that it would be useful for iden-tifying new deletions in the VWS region.

To screen for deletions that cause VWS, samplesfrom 37 unrelated propositi were genotyped withD1S3753. Samples from families VWS771, VWS1450,and VWS1473 were mono-allelic (data not shown). Totest whether these individuals were homozygous orhemizygous at D1S3753, additional members in thesefamilies were genotyped. Family VWS1473 was shownpreviously to carry a microdeletion [Sander et al.,1994]. Figure 1 shows that the affected children lack amaternal allele, indicating that the affected chromo-some is deleted at D1S3753. This result is consistentwith previous mapping data for the deletion in familyVWS1473 [Schutte et al., 1996]. In family VWS1450,the affected father is heterozygous for D1S3753, dem-onstrating that the disease-causing mutation in thisfamily is not a deletion that includes D1S3753 (datanot shown). In family VWS771, both the proposita andher affected mother lack an allele from their respectiveaffected parent, demonstrating an allele loss in the af-fected members of this family at D1S3753. As a controlfor sample error, nonpaternity and maternity, or isodi-somy, each of these families was genotyped with sevenmarkers outside of the VWS critical region (see Meth-ods). All control markers exhibited Mendelian inheri-tance. Therefore, the loss of allele detected in family771 is likely due to a deletion at chromosome bands1q32-q41.

Since D1S3753 efficiently detects deletions at 1q32-q41, we also screened other clefting populations for apossible disease-causing deletion. Popliteal pterygiumsyndrome (OMIM no. 119500) is an inherited cleftingdisorder that resembles VWS, but also includes web-bing of the lower limbs. Because of their similarity,VWS and PPS may be allelic. To test whether deletionsat 1q32-q41 contribute to the cause of PPS, we per-formed an allele loss assay with seven unrelated indi-viduals affected with PPS using D1S3753. All sevenindividuals with PPS were heterozygous for D1S3753,demonstrating that their phenotype is not associatedwith deletions near this marker.

TABLE II. Markers Used to Map Microdeletions in VWS Families

Marker D1S1663 D1S245 D1S471 D1S491 D1S70 D1S3754 GATA74A06 D1S3753 D1S205 yAS9R D1S414GDB ID no.a 685119 188425 199728 200285 438711 9798282 455934 9798272 187988 6544844 199114Polymorphismb STRP STRP STRP STRP STRP STRP STRP STRP STRP SSCP STRP

aThe source, primer sequences, and PCR conditions for each marker are available in GDB (www.gdb.org). Marker order is from CEN to TEL, and is basedon STS content mapping of a YAC contig (see Fig. 2).bPolymorphism is either short tandem repeat (STRP) or single-strand conformation (SSCP).

Microdeletions at 1q32-q41 That Cause VWS 147

Because the anomalies of VWS display wide expres-sivity, it is possible that clefts classified as nonsyn-dromic or syndromic with unknown cause may be mis-identified cases of VWS and may thus be caused bydeletions at 1q32-q41. To test this hypothesis, wescreened a panel of 178 samples from nonsyndromiccases and a panel of 76 samples from syndromic caseswhose etiology is unknown. In the nonsyndromic panel,30 individuals were mono-allelic, but none showed evi-dence of an allele loss. Similarly, in the syndromicpanel, five individuals were mono-allelic, but noneshowed evidence of an allele loss (data not shown).

Mapping of the 1q32-q41 Microdeletions

The phenotype of affected members of the VWS771family is confined to the classic traits of VWS. The

absence of other manifestations such as developmentaldelay in family VWS1473 [Sander et al., 1994] suggeststhat the microdeletion in family VWS771 may besmaller. To test this hypothesis, VWS771 andVWS1473 relatives were genotyped with other poly-morphic markers from the VWS region (Table II). Themicrodeletion in family VWS1473 was originally iden-tified from an allele loss at the marker D1S205 andsubsequently shown to include all the STSs fromD1S491 to D1S205 [Schutte et al., 1996]. Geneticanalysis of markers across the VWS region are consis-tent with the previous mapping data (Fig. 2). While theregion containing the distal end for both microdeletionsis similar, the proximal end of the deletion in familyVWS771 extends beyond the microdeletion in familyVWS1473 to include the markers D1S471 and D1S245.Surprisingly then, the deletion in family VWS771 maybe larger, even though affected members of familyVWS771 do not display developmental delay, nor anyother apparent anomaly that may be associated withthis deletion. Based on the size and STS content of YACclones that are derived from this region [Schutte et al.,1996], we estimate that the proximal breakpoint of themicrodeletion in family VWS771 extends 0.2 to 1.0 Mbbeyond the proximal end of the microdeletion in familyVWS1473 (Fig. 2). To test whether this deletion couldbe detected microscopically, cytogenetic analysis wasperformed on metaphase chromosomes derived from anaffected member of family VWS771. Both homologs ofchromosome 1 appeared normal (data not shown), in-dicating that a 1–2 Mb deletion could not be detectedmicroscopically from this region.

DISCUSSION

We report the identification of a large microdeletionat 1q32-q41 in affected members of family VWS771whose phenotype is confined to the cardinal findings ofVWS. Previously, two cases of VWS were shown to bedue to deletions at 1q32-q41. The first case was re-ported by Bocian and Walker [1987] and involved alarge interstitial deletion of chromosome 1q that couldbe detected microscopically. The second case involved asubmicroscopic deletion in family VWS1473 [Sander etal., 1994]. In both of the previous cases, the affected

Fig. 1. Genotypes of VWS families 1473 and 771. Pedigrees are shownabove the gels and the genotype for marker D1S3753 corresponds to thealleles displayed in the lane below each individual. Symbols in the pedi-grees represent affected (filled) and unaffected (open) individuals andmales (squares) and females (circles). The allele assignments within a fam-ily are indicated to the left of the gel.

Fig. 2. Physical map of microdeletions on 1q32-q41 that cause VWS. The VWS critical region (shaded) is based on previous recombinant and deletionmapping [Schutte et al., 1996]. Mapping of markers is based on STS content analysis of a previously described YAC contig [Schutte et al., 1996]. The sizeand addresses of CEPH YAC clones (horizontal bars), as well as, some of the STS content data were taken from that study. The microdeletions fromfamilies VWS1473 and VWS771 are represented by the shaded rectangles. The area between the parentheses represents the region containing thedeletion breakpoints.

148 Schutte et al.

individuals exhibited additional anomalies includingdevelopmental delay along with the classic findings ofVWS [Bocian and Walker, 1987; Sander et al., 1994].Surprisingly, while no additional anomalies were ob-served in the new microdeletion case, physical map-ping demonstrated that it spans 0.2–1.0 Mb beyond theproximal breakpoint of the microdeletion reported bySander et al. [1994]. Although the microdeletion iden-tified in family VWS771 did not refine the VWS criticalregion, its identification further supports the localiza-tion of the gene for this disorder to 1q32-q41 betweenthe markers D1S491 and D1S205. In addition, its pres-ence suggests that other, smaller microdeletions mayexist in our population of VWS families and their iden-tification would help localize the VWS gene.

The regions containing the distal breakpoints forboth microdeletions overlap considerably, suggesting apotential region of instability at chromosome 1q32-q41.Clustering of breakpoints is common in microdeletionsyndromes, and studies have shown that an importantunderlying mechanism for these observations is ho-mologous recombination between large duplicated se-quences [Nicholls et al., 1987; Dutly and Schinzel,1996; Reiter et al., 1996; Urban et al., 1996; Campbellet al., 1997; Carrozzo et al., 1997; Chen et al., 1997;Robinson et al., 1998]. Despite this potential cluster-ing, microdeletions at the VWS locus only account for5% (2/37) of the disease-causing mutations in our popu-lation. This relatively modest frequency suggests a lackof predisposing duplicated regions near the VWS locus.More extensive mapping and sequencing of this regionare required to determine the origin of the deletions, aswell as additional genetic studies to test for recombi-nation events.

The identification of microdeletions with D1S3753demonstrates the utility of this novel marker as a toolto identify large deletions at 1q32-q41 and suggeststhat it may now be possible to identify such microde-letions in new cases of Van der Woude syndrome. Thismarker should provide more effective carrier detectionin families that contain microdeletions, and assist inconfirming linkage to chromosome 1 in even smallfamilies. However, it is important to emphasize that todate there has been no evidence for linkage heteroge-neity and thereby locus heterogeneity in Van derWoude syndrome [Wienker et al., 1987; Murray et al.,1990; Sander et al., 1993, 1995; Bahuau et al., 1998].With this marker, it is also possible to screen for 1q32-q41 microdeletions in other cases of cleft lip and palate,both with and without lip pits that include additionalmultiple anomalies. This combination of anomaliesmay represent extensions of the region of the microde-letions reported here and result in haploinsufficiency ofadjacent genes. Although we examined 76 cases withcleft lip and palate and multiple anomalies for the pres-ence of a deletion, none were identified, but their exis-tence is nonetheless proposed.

The identification of a second VWS case caused by amicrodeletion also supports the hypothesis that VWS iscaused by haploinsufficiency. We can not be sure, atthis time, that the VWS gene itself is deleted, but re-combinant mapping [Schutte et al., 1996] and the iden-tification of two other deletions that cause VWS [Bo-

cian and Walker, 1987; Sander et al., 1994] are alsoconsistent with a mechanism of haploinsufficiency forthis disorder. As observed with other disorders causedby haploinsufficiency [Edwards et al., 1997; Prosserand van Heyningen, 1998], we expect to identify abroad spectrum of gene-inactivating mutations such asnonsense and splicing mutations in individuals af-fected with this disorder.

Finally, the most surprising observation is that 1–2Mb of DNA can be deleted and result only in a previ-ously well-described autosomal dominant Mendelianphenotype without any additional associated anoma-lies. Mapping studies (B. Bjork, B. Schutte, K. Cop-page, and J. Murray, manuscript in preparation) haveshown that at least 10 genes are hemizygous in indi-viduals carrying this microdeletion without an appar-ent consequence. This observation suggests that it willbe important to search for deletions of all sizes associ-ated with other autosomal dominant disorders, par-ticularly those that present as disorders of morphogen-esis and can result from haploinsufficiency of DNAtranscription factors or other genes now shown to befrequently involved in early embryogenesis. The loss ofthis very large amount of DNA with minimal effect ona phenotype outside of the single gene proposed to beinvolved in the disorder is almost without precedentand suggests that most human genes may be sufficientin a haploid state for the normal development of theorganism. The development of high-density, high-throughput genetic maps that are based on singlenucleotide polymorphisms (SNPs) will allow this hy-pothesis to be tested.

ACKNOWLEDGMENTS

We acknowledge Margaret Malik, Dee Even, CarlaNishimura, and Mindy Crall for technical help, andNancy Newkirk and Buck Huppman for administrativesupport. We thank the clinicians listed in the Methodssection for providing us with samples and informationfrom VWS families, and we particularly appreciate thecooperation of the families. This paper is dedicated toDr. Achim Sander who died in the course of its prepa-ration, and we are grateful to his sister, Helga Sander,for her continuing efforts to insure that his memory ishonored by research into the disorder for which Achimcontributed so much. This work was supported by NIHgrants P50-DE09170 (to J.C.M. and B.C.S.), R01-DE08559 (to J.C.M.), P30-HD27748 (Frank Morrissand B.C.S.) and Action Research grant S/P/3261(M.J.D.).

REFERENCES

Bahuau M, Houdayer C, Soupre V, Cougoureux E, Feldmann D, Aymard P,Munnich A, Martinez H, Lacombe D, Vazquez M-P. 1998. Linkageanalysis to 1q32 markers in 6 novel Van der Woude syndrome families.Am J Hum Genet 63:A280.

Bocian M, Walker AP. 1987. Lip pits and deletion 1q32-q41. Am J MedGenet 26:437–443.

Campbell L, Potter A, Ignatius J, Dubowitz V, Davies K. 1997. Genomicvariation and gene conversion in spinal muscular atrophy: implicationsfor disease process and clinical phenotype. Am J Hum Genet 61:40–50.

Carrozzo R, Rossi E, Christian SL, Kittikamron K, Livieri C, Corrias A,Pucci L, Fois A, Simi P, Bosio L, Beccaria L, Zuffardi O, Ledbetter DH.

Microdeletions at 1q32-q41 That Cause VWS 149

1997. Inter- and intrachromosomal rearrangements are both involvedin the origin of 15q11-q13 deletions in Prader-Willi syndrome. Am JHum Genet 61:228–231.

Chen KS, Manian P, Koeuth T, Potocki L, Zhao Q, Chinault AC, Lee CC,Lupski JR. 1997. Homologous recombination of a flanking repeat genecluster is a mechanism for a common contiguous gene deletion syn-drome. Nat Genet 17:154–163.

Chong SS, Pack SD, Roschke AV, Tanigami A, Carrozzo R, Smith AC,Dobyns WB, Ledbetter DH. 1997. A revision of the lissencephaly andMiller-Dieker syndrome critical regions in chromosome 17p13.3. HumMol Genet 6:147–155.

Dutly F, Schinzel A. 1996. Unequal interchromosomal rearrangementsmay result in elastin gene deletions causing the Williams-Beuren syn-drome. Hum Mol Genet 5:1893–1898.

Edwards SJ, Gladwin AJ, Dixon MJ. 1997. The mutational spectrum inTreacher Collins syndrome reveals a predominance of mutations thatcreate a premature-termination codon. Am J Hum Genet 60:515–524.

Francke U, Ochs HD, de Martinville B, Giacalone J, Lindgren V, DistecheC, Pagon RA, Hofker MH, van Ommen G-JB, Pearson PL, WedgwoodRJ. 1985. Minor Xp21 chromosome deletion in a male associated withexpression of Duchenne muscular dystrophy, chronic granulomatousdisease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet37:250–267.

Kazazian HH, Boehm CD. 1988. Molecular basis and prenatal diagnosis ofbeta-thalassemia. Blood 72:1107–1116.

Lidral AC, Romitti PA, Basart AM, Doetschman T, Leysens NJ, Daack-Hirsch S, Semina EV, Johnson LR, Machida J, Burds A, Parnell TJ,Rubenstein JLR, Murray JC. 1998. Association of MSX1 and TGFB3with nonsyndromic clefting in humans. Am J Hum Genet 63:557–568.

Miller SA, Dykes DD, Polesky HF. 1988. A simple salting out procedure forextracting DNA from human nucleated cells. Nucleic Acids Res 16:1215.

Munger RG, Romitti PA, Daack-Hirsch S, Burns TL, Murray JC, HansonJ. 1996. Maternal alcohol use and risk of orofacial cleft birth defects.Teratology 54:27–33.

Murray JC, Nishimura DY, Buetow KH, Ardinger HH, Spence MA,Sparkes RS, Falk RE, Falk PM, Gardner RJ, Harkness EM, Glinski LP,Pauli RM, Nakamura Y, Green PP, Schinzel A. 1990. Linkage of anautosomal dominant clefting syndrome (Van der Woude) to loci on chro-mosome 1q. Am J Hum Genet 46:486–491.

Nicholls RD, Fischel-Ghodsian N, Higgs DR. 1987. Recombination at thehuman alpha-globin gene cluster: sequence features and topologicalconstraints. Cell 49:369–378.

Nickerson E, Greenberg F, Keating MT, McCaskill C, Shaffer LG. 1995.Deletions of the elastin gene at 7q11.23 occur in approximately 90% ofpatients with Williams syndrome. Am J Hum Genet 56:1156–1161.

Ottolenghi S, Comi P, Giglioni B, Tolstoshev P, Lanyon WG, Mitchell GJ,Williamson R, Russo G, Musumeci S, Schillro G, Tsistrakis GA, Char-ache S, Wood WG, Clegg JB, Weatherall DJ. 1976. Delta-beta-thalassemia is due to a gene deletion. Cell 9:71–80.

Ottolenghi S, Lanyon WG, Paul J, Williamson R, Weatherall DJ, Clegg JB,Pritchard J, Pootrakul S, Boon WH. 1974. The severe form of alphathalasaemia is caused by a haemoglobin gene deletion. Nature 251:389–392.

Prosser J, van Heyningen V. 1998. PAX6 mutations reviewed. Hum Mutat11:93–108.

Ramirez F, O’Donnell JV, Marks PA, Bank A, Musumeci S, Schiliro G,Pizzarelli G, Russo G, Luppis B, Gambino R. 1976. Abnormal or absentbeta mRNA in betao Ferrara and gene deletion in delta beta thalas-saemia. Nature 263:471–475.

Reiter LT, Murakami T, Koeuth T, Pentao L, Muzny DM, Gibbs RA, LupskiJR. 1996. A recombination hotspot responsible for two inherited pe-ripheral neuropathies is located near a mariner transposon-like ele-ment. Nat Genet 12:288–297.

Robinson WP, Dutly F, Nicholls RD, Bernasconi F, Penaherrera M, Micha-elis RC, Abeliovich D, Schinzel AA. 1998. The mechanisms involved information of deletions and duplications of 15q11-q13. J Med Genet35:130–136.

Sander A, Moser H, Liechti-Gallati S, Grimm T, Zingg M, Raveh J. 1993.Linkage of Van der Woude syndrome (VWS) to REN and exclusion ofthe candidate gene TGFB2 from the disease locus in a large pedigree.Hum Genet 91:55–62.

Sander A, Murray JC, Scherpbier-Heddema T, Buetow KH, WeissenbachJ, Zingg M, Ludwig K, Schmelzle R. 1995. Microsatellite-based finemapping of the Van der Woude syndrome locus to an interval of 4.1 cMbetween D1S245 and D1S414. Am J Hum Genet 56:310–318.

Sander A, Schmelzle R, Murray J. 1994. Evidence for a microdeletion in1q32-41 involving the gene responsible for Van der Woude syndrome.Hum Mol Genet 3:575–578.

Schutte BC, Sander A, Malik M, Murray JC. 1996. Refinement of the Vander Woude gene location and construction of a 3.5-Mb YAC contig andSTS map spanning the critical region in 1q32-q41. Genomics 36:507–514.

Taylor JM, Dozy A, Kan YW, Varmus HE, Lie-Injo LE, Ganesan J, Todd D.1974. Genetic lesion in homozygous alpha thalassaemia (hydrops feta-lis). Nature 251:392–393.

Urban Z, Helms C, Fekete G, Csiszar K, Bonnet D, Munnich A, Donis-Keller H, Boyd CD. 1996. 7q11.23 Deletions in Williams syndromearise as a consequence of unequal meiotic crossover. Am J Hum Genet59:958–962.

Wienker TF, Hudek G, Bissbort S, Mayerova A, Mauff G, Bender K. 1987.Linkage studies in a pedigree with Van der Woude syndrome. J MedGenet 24:160–162.

150 Schutte et al.