High Resolution Characterization of an InterstitialDeletion of Less Than 1.9 Mb at 4p16.3 AssociatedWith Wolf-Hirschhorn Syndrome

Y.-Y. Fang,1 S. Bain,1 E.A. Haan,2 H.J. Eyre,1 M. MacDonald,3 T.J. Wright,4 M.R. Altherr,4 O. Riess,5G. Sutherland,1 and D.F. Callen1*1Center for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital,North Adelaide, Australia

2Center for Medical Genetics, Department of Medical Genetics, Women’s and Children’s Hospital,North Adelaide, Australia

3Molecular Neurogenetics Laboratory, Massachusetts General Hospital and Department of Genetics, Harvard MedicalSchool, Boston, Massachusetts

4Life Science Division, Los Alamos National Laboratory, New Mexico5Department of Molecular Human Genetics, Ruhr-University, Bochum, Germany

Wolf-Hirschhorn syndrome (WHS) causedby 4p16.3 deletions comprises growth andmental retardation, distinct facial appear-ance and seizures. This study characterizeda subtle interstitial deletion of 4p16.3 in agirl with mild retardation and possessing fa-cial traits characteristic of WHS. The pa-tient had generalized seizures in conjunc-tion with fever at 3 and 5 years of age. Fluo-rescence in situ hybridization (FISH) with aseries of markers in the 4p16.3 regionshowed that the interstitial deletion in thispatient was between the probes D4S96 andD4S182, enabling the size of the deletion tobe estimated as less than 1.9 Mb. This is thesmallest interstitial deletion of 4p16.3 whichhas been reported. The patient contributesto a refinement of the phenotypic map of theWHS region in 4p16.3. The critical region forthe characteristic facial changes of WHS,failure to thrive and developmental delay isnow localized to a region of less than 700 kb.The mental retardation of this patient wasmild suggesting that small interstitial dele-tion may have less severe phenotypic conse-quences. Am. J. Med. Genet. 71:453–457,1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: Wolf-Hirschhorn syndrome;4p deletion; fluorescence insitu hybridization

INTRODUCTIONWolf-Hirschhorn syndrome (WHS) was indepen-

dently described by Wolf and Hirschhorn in 1965 [Wolfet al., 1965; Hirschhorn et al., 1965] and has an inci-dence of 1/50,000 live births [Johnson et al., 1976;Goodman and Gorlin, 1983]. It is characterized by se-vere growth and mental retardation, distinct facialanomalies and seizures. It is due to deletions of thedistal short arm of chromosome 4.

The deletions in the short arm of chromosome 4 canrange from one-half of the short arm [Lurie et al., 1980;Wilson et al., 1981] to cytogenetically undetectable[Preus et al., 1985]. These undetectable cases can beeither small deletions or cryptic translocations whichcan be detected by fluorescence in situ hybridiza-tion (FISH) [Altherr et al., 1991; El-Rifai et al., 1995] orpolymerase chain reaction (PCR) [Altherr et al.,1992]. The critical deletion region of WHS within4p16.3 was estimated to be 2 Mb between the probesD4S142 and D4S43 [Gandelman et al., 1992; Es-tabrooks et al., 1992]. Subsequently, Reid et al. [1996]excluded the loci D4S111 and D4S115 from the criti-cal deletion region and Somer et al. [1995] reported apatient with typical WHS who was not deleted forD4S96; therefore, the critical region was further re-duced to 1.2 Mb between the probes D4S96 and D4S43.The present study characterized a subtle interstitialdeletion of 4p16.3 in a girl possessing mild WHS mani-festations. Analysis with FISH maps this deletion tothe critical WHS region. The interstitial deletion inthis patient is estimated to be approximately 1.9 Mb insize.

Contract grant sponsor: National Health and Medical ResearchCouncil of Australia; Contract grant sponsor: Women’s and Chil-dren’s Hospital Research Foundation; Contract grant sponsor:U.S. Department of Energy; Contract grant number: W-7405-ENG-36; Contract grant sponsor: Los Alamos National Labora-tory Directed Research and Development Fund.

*Correspondence to: Dr. David F. Callen, Department of Cyto-genetics and Molecular Genetics, Women’s and Children’s Hospi-tal, North Adelaide SA 5006, Australia.

Received 21 October 1996; Accepted 3 March 1997

American Journal of Medical Genetics 71:453–457 (1997)

© 1997 Wiley-Liss, Inc.

MATERIALS AND METHODSClinical Report

KS was the first child of Caucasian parents, mater-nal age 18 years and paternal age 25 years. Birth wasat 32 weeks by emergency Caesarean section becauseof antepartum haemorrhage. Apgar scores were 4 at 1minute and 7 at 5 minutes. Birth weight was 1,480 g(25th centile) and head circumference (OFC) was 28 cm(10th centile). She required ventilation for hyalinemembrane disease and phototherapy for jaundice.

She fed poorly, had gastro-oesophageal reflux andfailed to thrive, resulting in gavage feeding between 12and 15 months. Development was noted to be delayedin the first months of life and there were minor facialanomalies. Chromosomes were initially reported to benormal. Ultrasound study showed mildly dilated ven-tricles of the brain. There were recurrent respiratoryinfections during infancy. Immunoglobulin A defi-ciency could be demonstrated until age 2 years. Gen-eralized seizures occurred with fever at age 3 and 5years.

She was reviewed at 5.5 years. There was moder-ately severe developmental delay. Height, weight andhead circumference were well below, but tracking par-allel, to the 3rd centile. Mental retardation was mild.She had minor anomalies of ear shape, upslanting pal-pebral fissures, telecanthus, broad nasal bridge, rela-tively short philtrum, small mouth, and small chin(Fig. 1). There was mild clinodactyly of the right littlefinger and broad halluces. There was an exaggeratedlumbar lordosis. Chromosome analysis was repeatedbecause the craniofacial changes suggested a diagnosisof Wolf-Hirschhorn syndrome.

Cytogenetic Analysis and FISH Study

High resolution cytogenetic studies were performedon the patient and her mother using trypsin-Giemsa(GTG) banding. The patient’s father was not availablefor study.

Molecular cytogenetic study of the patient was per-formed by FISH with markers located at 4p16.3, in-cluding D4S142 (2R88), D4F26 (pC847.351), D4S90(CD2), D4S133 (cDp16), D4S96 (pC678), D4S168

(8C10E4), D4S113 (A62.5), D4S98 (pC385.12), D4S166(L6), D4S43 (C9A), L25G12, 79F5, L65C1, D4S182(247F6), D4S127 (195C9), and D4S180 (21F12). Therelative order and distance between probes is given inFigure 4. A cosmid DNA, 77G3, located at 4q25 wasused as a control probe for chromosome 4. The FISHprocedures were used as described by Callen et al.[1990]. A fluorescein isothiocyanate-labeled avidin/antibody step was used to amplify hybridization signal.Cosmid DNA biotin labelled by nick translation werehybridized to metaphase spreads from the patient andher mother. Counterstaining of the propidium iodide-stained chromosomes with DAPI allowed identificationof chromosomes and location of signal to chromosomebands. Images of metaphase preparations were re-corded on color slides.

RESULTS

High resolution studies suggested a possible deletionof 4p16.3 but this could be observed only in some pro-metaphase spreads (Fig. 2). This deletion was con-firmed by FISH using the DNA probes, D4S168(8C10E4), D4S113 (A62.5), D4S98 (pC385.12), D4S166(L6), D4S43 (C9A), L25G12, 79F5, L65C1 since a hy-bridization signal from 4p16.3 region was detected onlyon one chromosome 4, and was missing from its homo-log (Fig. 3A). FISH with probes, D4S142 (2R88), D4F26(pC847.351), D4S90 (CD2), D4S133 (cDp16), D4S96(pC678), D4S182 (247F6), D4S127 (195C9), andD4S180 (21F12) showed a hybridization signal on bothchromosomes 4 (Fig. 3B). Therefore, there was an in-terstitial deletion of 4p16.3. The proximal breakpointwas between L65C1 and D4S182 (247F6), which areapproximately 3.2–3.4 Mb from the telomere, and thedistal breakpoint between D4S96 (pC678) and D4S168(8C10E4), which are approximately 1.5–2.06 Mb fromthe telomere. Since the deletion spans from D4S96/D4S168 to L65C1/D4S182, this patient has an intersti-tial deletion up to 1.9 Mb in size.

Lymphocyte cultures from the patient’s mothershowed a normal karyotype, and FISH studies with theprobes D4S43 (C9A) and D4S168 (8C10E4), which arelocated in the critical region of Wolf-Hirschhorn syn-drome at 4p16.3, were normal.

DISCUSSION

Molecular analysis of various WHS patients localizedthe critical region to an approximate 2-Mb interval be-tween D4S43 and D4S142 [Gandelman et al., 1992; Es-tabrooks et al., 1992]. Subsequently, molecular charac-terizations of several patients have reduced this criti-cal region. Reid et al. [1996] excluded the loci D4S111and D4S115 while Somer et al. [1995] excluded D4S96.In addition, Wright et al. [1996] described a WHS pa-tient with a distal breakpoint between D4S168 andFGFR3. Therefore, the critical region of WHS is nowbetween D4S168/FGFR3 and D4S166/D4S43 whichspans an interval of 450–700 kb (Fig. 4). The patient,KS, in this report is consistent with this critical regionFig. 1. Photographs of patient at age 5.5 years.

454 Fang et al.

for WHS and confirms that the loci D4S111, D4S115,and D4S96 are outside the critical region. The probeD4S96 should be used with caution for the FISH diag-nosis of WHS since it is outside the critical region andtherefore could lead to false negative results.

Another closely related syndrome is Pitt-Rogers-Danks syndrome (PRD). Sixteen cases with PRD havebeen reported [Donnai, 1986, 1996; Oorthuys andBleeker-Wagemakers, 1989; Lizcano-Gil et al., 1995;Clemens et al., 1996; Lindeman-Kusse et al., 1996; Zol-lino et al., 1996; and De Die-Smulders and Engelen,1996] since it was initially described by Pitt et al. in1984. The phenotype in PRD partially overlaps withthat of WHS with mental, growth retardation, and sei-zures in common, while craniofacial features of PRD,including the wide mouth, short upper lip with flatphiltrum, beak nose, maxillary hypoplasia, prominenteyes, and palmar creases have not been reported inWHS. The detailed comparison of these two syndromeshas been described [Clements et al., 1996; Lindeman-

Kusse et al., 1996]. FISH with the probe D4S96 showsthat 9 of 16 PRD patients have a 4p deletion. The pro-portion of PRD patients with established deletions maybe an underestimate since not all patients were studiedwith FISH and where FISH was used the probe may beoutside the critical region. Whether these two syn-dromes are different clinical presentations of the samebasic defect or are the result of deletions involving dif-ferent, but overlapping, critical region has yet to beestablished. Although the abnormality of our patient ismild, which is usually suggestive of PRD, she does notpossess the striking facial appearance of maxillary hy-poplasia, wide mouth, and ocular proptosis that arecharacteristics of PRD.

The physical distances given in Figure 4 are derivedfrom several sources [Whaley et al., 1988, 1991; Snellet al., 1992; McCombie et al., 1992; Collins et al.,1992b; Baxendale et al., 1993]. The patient KS of thisreport has a deletion less than 1.9 Mb in size fromD4S96/D4S168 to L65C1/D4S182, which is the small-

Fig. 2. The deletion of chromosome 4 is indicated in the G-banded partial metaphase by the arrow.

Fig. 3. Partial metaphases after in situ hybridization with 4p probes. A: Probe D4S166, signal can be seen on the normal chromosome 4 (solid arrow)but not on the other partially deleted chromosome 4 (open arrow). The chromosome 4s was additionally indicated by the cosmid 77G3 which is locatedon 4q25. B: Probe D4S127 was present (arrows) on both chromosome 4s and is therefore not deleted. The chromosomes were identified by DistamycinA/DAPI banding (not presented).

4p Deletion in Wolf-Hirschhorn Syndrome 455

est interstitial deletion yet reported in a WHS patient.This is consistent with the experience of clinical cyto-genetic analysis, where the lower limit of detection ofdeletions are in the order of two megabases in size[Ledbetter and Ballabio, 1995].

So far, there are 6 known expressed genes in the4p16.3 region, including a zinc-finger gene (ZNF141)[Tommerup et al., 1993], b-subunit of rod cGMP phos-phodiesterase (PDE6B) [Collins et al., 1992a], myosinlight chain (MYL5) [Collins et al., 1992b], a-L-iduronidase (IDUA) [Scott et al., 1990], HDA1-1 [Mc-Combie et al., 1992], and fibroblast growth factor re-ceptor 3 (FGFR3) [Thompson et al., 1991]. Since thefirst four genes are located distal to the critical region,they are unlikely to be the candidate genes in the de-velopment of this syndrome. FGFR3 and HDA1-1, onthe other hand, are possible candidate genes for thefollowing reasons. FGFR3, which was isolated byKeegan et al. [1991], has been shown to be responsiblefor achondroplasia [Rousseau et al., 1994; Shiang et al.,1994]. If FGFR3 contributes to WHS, it is unlikely toact alone since the phenotype in WHS involves morethan one organ. HDA1-1 may also contribute to theWHS phenotype since it was deleted in patient KS andlocated at the distal boundary of the patient MS (Fig.4). HDA1-1 gene is of unknown function and is ex-pressed in a variety of organs such as the brain, heart,

kidney, placenta [McCombie et al., 1992]. However,4p16.3 is a gene rich region and several expressed se-quences have now been identified by sequencing theregion spanning D4S43 and D4S98 by Sanger Centre(see http://www.sanger.ac.uk).

The phenotype of this presented patient is broadlyconsistent with the phenotypic map of Estabrooks et al.[1995]. An exception is slanting palpebral fissures,which were present in the patient KS but mapped to amore distal region by Estabrooks et al. [1995]. Renalabnormality, hearing loss, and midline defects such asfused teeth and congenital heart defect are not presentin our patient and are consistent with larger deletionsextending proximal to D4S43. The typical physiognomyand microcephaly are now localized on the phenotypicmap to a maximum interval of 700 kb. Although themental retardation of patient KS was mild, the severityof growth and mental retardation is relatively poorlycorrelated with deletion size in WHS patients [Es-tabrooks et al., 1995]. Patient KS was found to havetransient IgA deficiency which has not been previouslyreported. This may be the cause of early respiratoryinfection frequently encountered in WHS patients.

In conclusion, the patient has the smallest, intersti-tial deletion in WHS yet reported and is consistentwith the critical region of WHS being a maximum sizeof 700 kb.

ACKNOWLEDGMENTS

This research was supported by the National Healthand Medical Research Council of Australia and Wom-en’s and Children’s Hospital Research Foundation inAdelaide, South Australia. Funding was provided byUS Department & Energy under contract [W-7405-ENG-36 (MRA) and the Los Alamos National Labora-tory Directed Research and Development Fund (TW,MRA).

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