Molecular and genomic characterisation of cryptic chromosomal alterations leading to paternal duplication of the 11p15.5 Beckwith‐Wiedemann region

Abstract

Background

Beckwith‐Wiedemann syndrome (BWS) is an overgrowth disorder with increased risk of paediatric tumours. The aetiology involves epigenetic and genetic alterations affecting the 11p15 region, methylation of the differentially methylated DMR2 region being the most common defect, while less frequent aetiologies include mosaic paternal 11p uniparental disomy (11patUPD), maternally inherited mutations of the CDKN1C gene, and hypermethylation of DMR1. A few patients have cytogenetic abnormalities involving 11p15.5.

Methods

Screening of 70 trios of BWS probands for 11p mosaic paternal UPD and for cryptic cytogenetic rearrangements using microsatellite segregation analysis identified a profile compatible with paternal 11p15 duplication in two patients.

Results

Fluorescence in situ hybridisation analysis revealed in one case the unbalanced translocation der(21)t(11;21)(p15.4;q22.3) originated from missegregation of a cryptic paternal balanced translocation. The second patient, trisomic for D11S1318, carried a small de novo dup(11)(p15.5p15.5), resulting from unequal recombination at paternal meiosis I. The duplicated region involves only IC1 and spares IC2/LIT1, as shown by fluorescent in situ hybridisation (FISH) mapping of the proximal duplication breakpoint within the amino‐terminal part of KvLQT1.

Conclusions

An additional patient with Wolf‐Hirschorn syndrome was shown by FISH studies to carry a der(4)t(4;11)(p16.3;p15.4), contributed by a balanced translocation father. Interestingly, refined breakpoint mapping on 11p and the critical regions on the partner 21q and 4p chromosomal regions suggested that both translocations affecting 11p15.4 are mediated by segmental duplications. These findings of chromosomal rearrangements affecting 11p15.5–15.4 provide a tool to further dissect the genomics of the BWS region and the pathogenesis of this imprinting disorder.

Beckwith‐Wiedemann syndrome (BWS; MIM 130650) is a heterogeneous overgrowth disorder associated with an increased risk (7.5%) of developing embryonal abdominal tumours including Wilms tumour, adrenocortical carcinomas, and hepatoblastoma.¹,² No consensus diagnostic criteria for BWS exist; it is accepted that a diagnosis is based on the presence of at least three major findings or two major and one minor finding. Macrosomia, macroglossia, omphalocele/umbilical hernia, hemihypertrophia, embryonal tumours, adrenocortical cytomegaly, renal anomalies, ear lobe creases/posterior helical ear pits, and cleft palate (rare) are major findings associated with BWS.³,⁴,⁵ Neonatal hypoglycaemia, polyhydramnios or enlarged placenta, a characteristic facies with midfacial hypoplasia, advanced bone age, cardiomegaly or structural cardiac anomalies, facial nevus flammeus, and haemangioma are minor findings. Most BWS children have normal intelligence and grow to an adult height within the normal range. Presentation is mainly sporadic with 15% of cases being familial.⁶

The aetiology of BWS is complex and involves genetic and epigenetic mechanisms within the 11p15 chromosomal region. Up to 60% of sporadic cases are due to epigenetic modifications involving two distinct imprinting centres, IC1 and IC2, regulating imprinted gene expression over a long distance.⁷,⁸ IC1 consists of a differentially methylated region (DMR1) positioned upstream of H19 and containing target sites for the insulator protein CTCF, which regulates access to the same enhancers of the two reciprocally imprinted genes H19 and IGF2.⁹ Methylation on the paternal allele prevents CTCF from binding DMR1, permitting expression of IGF2; on the unmethylated maternal allele, insulator protein binding does not allow IGF2 promoters to interact with the enhancers downstream of H19.⁹,¹⁰ Gain of DMR1 methylation leading to overexpression of IGF2 and absence of H19 transcript is found in 5–10% of sporadic BWS cases.¹¹,¹² The IC2 specific DMR (KvDMR) is located in intron 10 of the maternally expressed KCNQ1. On the paternal unmethylated allele, an antisense transcript within intron 10, KCNQ1OT1 (or LIT1) overlaps KvDMR and acts in cis silencing the KCNQ1 and CDKN1C genes; conversely, on the maternal methylated DMR2 the two genes are expressed. Hypomethylation of IC2 leading to loss of methylation at the KvDMR is seen in up to 50% of sporadic BWS cases.¹¹,¹²,¹³,¹⁴

Regarding the genetic mechanisms responsible for BWS, segmental paternal disomy of 11p15 is recorded at a frequency of 10–20%¹⁵; mutations in the coding sequence of the cyclin kinase dependent inhibitor p57 are involved in 5% of patients,¹⁶ rising to 40% in familial cases.¹⁷

Germline chromosomal rearrangements involved in BWS occur very rarely and include maternally and paternally derived translocations, inversions, or duplication of the 11p15 region.⁶ The maternally inherited translocations and inversions are always balanced and their breakpoints cluster to three regions of 11p15: the most distal breakpoint region is 400 kb proximal to IGF2, the second is proximal to the beta‐haemoglobin gene (HBB), and the third clusters within the KCNQ1 gene, altering imprinting of KCNQ1OT1.¹⁸ Duplication of chromosomal region 11p13–p15 responsible for BWS most frequently results from unbalanced segregation of a paternal translocation,¹⁹ but de novo duplications of the paternally derived 11p15 region were also found.¹⁹

A molecular search of 11patUPD performed on a cohort of 70 Italian BWS patients, overall representing the heterogeneous clinical spectrum of the syndrome, allowed us to identify two patients with cryptic chromosomal rearrangements leading to disomy of 11p15 on the paternal chromosome. The patients clinically had classic BWS, carrying an unbalanced t(11;21) translocation and an interstitial 11p15 duplication, respectively. A third case, clinically diagnosed as Wolf‐Hirschorn syndrome (WHS), was also included following detection of an unbalanced cryptic t(4;11) translocation. We herein report on clinical and fluorescent in situ hybridisation (FISH) characterisation allowing the extent and boundaries of the duplicated region on paternal 11p15 to be defined in all three cases. Refined FISH mapping of translocation and duplication breakpoints suggested that genomic motifs of homology mediated the inter‐ and intrachromosomal rearrangements, consistent with the well known genomic instability of the 11p15.5 region.

Methods

Microsatellite analysis

DNA was extracted from probands' and parents' peripheral blood leukocytes using a commercial Nucleon DNA Extraction Kit (Amersham Bioscience, Little Chalfont, UK).

For microsatellite segregation analysis, PCR analysis of all markers, except D11S4046 and D11S1338, was performed in the first family using γATP³² labelled primers, which sequences were downloaded from the GenBank electronic database; details of PCR and polyacrylamide gel electrophoresis conditions have been previously reported.²⁰ For cases 2 and 3, PCR employed fluorescinated primers, selected from the ABI Prism Linkage Mapping Set for chromosome 11 and analysis was performed on the automated ABI 310 sequencer. PCR cycling, pool dilution, and capillary electrophoresis have been specified previously. In both protocols, the number of PCR cycles was adjusted to stop the reaction before the plateau phase to allow even low grade pat11UPD mosaicism to be detected. Normalisation of the ratio between allele areas was performed at the probands' heterozygous loci as described previously.²⁰

Table 1 Growth parameters at birth and at diagnosis for all patients.

Clinical features shown by BWS patients				Clinical features shown by WHS patient (Pt3)
A	Patient 1	Deceased brother of patient 1	Patient 2	B	Patient 3
Measures at birth				Measures at birth
Length	53 cm (90°p)	58 cm (>97°p)	47.5 cm (<50°p)	Length	Probably low for gestational age
Weight	4250 g (>97°p)	5100 g (>97°p)	4360 g (>97°p)	Weight	Probably low for gestational age
CC	34.5 cm (3–10°p))	NK	37 cm (<3°p)	CC	Probably low for gestational age
Apgar index	3, 5	7	8, 9	Apgar index	Probably low (asphyxia/fetal distress)
Age at diagnosis	2 months	At birth	6 years	Age at diagnosis	15 years
Measures at diagnosis				Measures at diagnosis
Length	56 cm (50°p)	58 cm (>97°p)	116 cm (75°p)	Length	160 cm (10°p)
Weight	4870 g (75°p)	5100 g (>97°p)	25.9 kg (∼97°p)	Weight	39.5 kg (<3°p)
CC	36 (3°p)	NK	52 cm (75°p)	CC	51 cm (<3°p)
Major BWS features				WHS clinical features
Macrosomy	+	+	−	Typical craniofacial features	+
Macroglossia	+	+	+	Prenatal onset growth deficiency	NK, probably low for gestational age
Omphalocele	−	+	−	Postnatal growth retardation,	+
				including microcephaly
Embryonal tumour	−	Nephroblastoma	−	Hypotonia	+
Hemihypertrophy	−	−	+	Developmental delay and MR	Severe with absent language
Renal abnormalities	Hydronephrosis at birth,	−	−	Seizures (50–100%)	Generalised tonic‐clonic seizures
	polycystic kidney
Minor BWS features				Skeletal anomalies (60–70%)	Thoracolumbar scoliosis
Polyhydramnios	−	−	+	Congenital heart defects (30–50%)	−
Haemangioma	−	−	+	Cleft lip and cleft palate (30–50%)	−
Diastasis recti	+	+	+	Colobomata (30–50%)	−
Developmental delay	Borderline/mild	/	−	Hypospadias (30–50%)	−
Learning disabilities	−	/	+
Additional features				Urinary tract malformations (25%)	−
Hypotonia and	+	+	+	Hearing loss (>40%)	−
hyporeactivity
Seizures, brain oedema,	−	+	−	Structural brain abnormalities (33%)	Hypoplasia of corpus
adrenal haemorrhage,					callosum, absence of forceps
pneumothorax					major and gyrus cinguli
Cryptorchidism	Monolateral	Bilateral	/	Other features reported in WHS	Umbilical hernia, right
Scoliosis	−	/	+		preauricular tag

In section A, major and minor BWS features, as well as additional findings shown by BWS patients are displayed to make the comparison easier. In section B, all WHS features are compared to those shown by patient 3. Percentages are displayed only for non‐mandatory findings (NK, not known). CC, cranial circumference; °p, percentile. MR, mental retardation.

H19 methylation analysis

H19 methylation analysis targeted the upstream gene region by using the methylation sensitive HpaII enzyme and HhaI RFLPs. Genomic DNAs from the probands and their parents were amplified using primers II and III as indicated.²¹ A fifth of the 435 bp PCR product was then digested by 12 U of HhaI (New England Biolabs, Ipswich, MA). The heterozygous pattern is evidenced by the presence of three bands of 435, 245, and 190 bp. Following genomic HpaII digestion, PCR by primers II and III only amplifies the paternal methylated allele, which is discriminated by HhaI digestion.

LIT1 methylation analysis

Genomic DNA was digested by BamHI and the methylation sensitive NotI enzyme according to the published protocol.²² Filters were hybridised to a KCNQ1OT1 probe obtained by purification of the amplified PCR product.¹¹ Filters were washed for 20 min in 2× SSC/0.1% SDS at room temperature, for 15 min in 0.5× SSC/0.1% SDS at 60°C, and for 10 min in 0.1× SSC at 65°C.

Karyotyping and FISH characterisation

Chromosomes were prepared from peripheral blood lymphocyte cultures following standard procedures. FISH with chromosome specific subtelomeric probes was performed according to the manufacturer's specifications (Cytocell, Oxford, UK). The BAC probes used in the FISH study are listed in the online supplementary table 2 available at http://www.jmedgenet.com/supplemental. The RPCI‐11 BAC clones and the clones derived from the “CalTech” Human BAC Libraries (CTD), localised within 11p15.5–15.4, 4p16.3–4p16.2, and 21q22.3, were selected according to the UCSC Genome Browser (May 2004 release).

All the probes were nick translation labelled with biotin, digoxigenin (La Roche, Basel, Switzerland), or CY3‐dUTP (Amersham Biosciences). The FISH protocols of Lichter²³ and Lichter and Cremer²⁴ were followed with minor modifications.

All parents signed an appropriate consent form.

Results

Clinical reports

Proband 1, born at 37 weeks' gestation, to unrelated parents, is the youngest male in a sibship including two first trimester miscarriages and a brother (III:3) who died 1 month after birth. As detailed in table 1A, BWS diagnosis in both sibs was based on the presence of at least three major criteria. All the clinical signs displayed at birth by the proband (table 1A) were previously observed in the deceased brother, who is shown in fig 1A. III:3 also presented with nephroblastoma, omphalocele, adrenal haemorrhages, cerebral oedema and haemorrhages, seizures, and pneumothorax (table 1A), which together were responsible for his death.

Figure 1 Family pedigrees and pictures of the three patients. (A) The family pedigree indicates familial recurrence and miscarriages; the arrow points to proband 1 (III:7). A picture of the proband's deceased brother is shown below the family tree. (B) The family pedigree of the sporadic case of proband 2 is shown and, below, a photograph showing the dismorphic features of proband 2. (C) The family tree of the WHS proband shows indicates a familial case, while, below, a picture of proband 3 in childhood is suggestive for the clinical phenotype. (Patient photographs are reproduced with consent.)

Proband 2 is a female patient, born at 38 weeks' gestation to unrelated parents with an unremarkable family history. Labour was induced because of polyhydramnios, and the patient was delivered by caesarean section. She needed nasogastric feeds for the first 5 days after birth and required physiotherapy until 14 months of age to improve hypotonia. BWS diagnosis was based on the presence of two major BWS features (macroglossia and slight right hemihypertrophy of face and lower limb) plus a few minor signs such as diastasis recti and a large tuberous‐cavernous haemangioma at the right thorax (table 1A). Her facial dysmorphisms, including a webbed and short neck, are shown in fig 1B. Additional non‐particular BWS signs, such as a high and narrow palate, severe lordosis, and a worsening cervicothorax and thoracolumbar scoliosis were also present. Her IQ was within normal range at 5 years, when she began speech therapy.

Proband 3, the first son of unrelated and healthy parents, was born at 38 weeks' gestation. After birth he developed respiratory distress and briefly had hypoglycaemia; subsequent growth was poor and he continued to have central hypotonia. Developmental delay was apparent at 6 months of age. Brain MRI, conducted at the age of 3, showed hypoplasia of corpus callosum and absence of both forceps major and gyrus cinguli. He developed epilepsy with generalised tonic‐clonic seizures at approximately 5 years of age. He began to walk between 8 and 9 years of age. His clinical features, summarised in table 1B, were suggestive of WHS. Figure 1C shows the WHS phenotype with broad forehead, prominent glabella, hypertelorism, arched eyebrows, short philtrum, and micrognathia. The family pedigree (fig 1C) shows an uncle on the father's side (II:2) with mental retardation and two first trimester miscarriages (III:2 and III:3).

Microsatellite analysis

Segregation analysis from parents to probands of microsatellites mapping to the BWS 11p15 region (D11S1363, D11S922, D11S4046, TH, D11S1318, D11S4146, D11S1338, D11S1760, and D11S1331) and outside D11S935 (11p12–p13) was performed for the two BWS patients to detect segmental paternal 11p disomy or chromosomal micro‐rearrangements leading to imbalance of this region. Figure 2 shows for each BWS patient the autoradiograms/electropherograms of the loci informative for segmental disomy, which are positioned on the map in tel‐cen order. In all three probands, segmental trisomy 11p was demonstrated by the finding of one maternal and both paternal alleles at a few investigated markers, allowing 11patUPD mosaicism to be ruled out. The D11S1331 locus was found to fix the centromeric limit of the trisomic region in proband 1 (fig 2A), while D11S4146 defined the centromeric boundary of rearrangements of both probands 2 and 3 (fig 2B,C). These findings, together with the records in the families of probands 1 and 3 of two subsequent interrupted pregnancies and a first/second grade relative with a similar phenotype, were strongly suggestive of an unbalanced cryptic translocation malsegregating from the father to the affected sib.

Figure 2 Segregation of 11p15.5–p15.4 microsatellites from parents to probands. Autoradiograms and electropherograms of the informative markers tested in the region for case 1 (A), case 2 (B), and case 3 (C) are shown (F, father; P, proband; M, mother). The occurrence of three alleles at D11S922 and D11S4046 is indicated as well as a double dosage (as measured by densitometry) of the paternal allele at D11S1363 and TH in patient 1; a trisomic pattern at locus D11S1318 and increased dosage of the paternal allele at the telomeric markers D11S1363, D11S4046, and TH is shown for case 2; and a trisomic pattern for D11S4046 and D11S1318 and a duplicated area at the paternal allele of the D11S1363 and TH loci is shown for case 3. The arrows indicate those microsatellites informative for the presence of a paternal duplication. A bar representing the relative position of the investigated microsatellites on the 11p15 chromosomal region is shown at the top of panel B.

FISH characterisation

Patient 1

FISH with subtelomeric probes revealed on the proband's metaphases the presence of an unbalanced der(21)t(11;21)(p15.4;q22.3) translocation leading to distal 21q monosomy and distal 11p trisomy. Parental FISH analysis showed that the derivative chromosome of the patient originated from malsegregation of a paternal balanced translocation t(11;21)(p15.4;q22.3).

FISH with BAC clones mapping within the 11p15.5–15.4 and 21q22.3 genomic intervals finely localised the translocation breakpoints. Namely, the 11pter breakpoint was mapped in the genomic region targeted by clone RP11‐348A20 (figs 3A and 4; see also online supplementary table 2), while the 21qter breakpoint was localised between RP11‐691J8 and RP11‐1F8 (fig 3B,C; also online supplementary table 2 and fig 6, both of which are available at http://www.jmedgenet.com/supplemental). BAC FISH characterisation established that the 11p duplication spans 3.8 Mb and the 21q deletion extends for 3.4 Mb.

Figure 3 Characterisation by BAC FISH analysis of the extent and boundaries of the 11p15 region duplicated in the three patients under study. (A, B, C) Partial metaphases of patient 1. FISH with BAC RP11‐348A20 (11p15.4) (A) shows an additional signal on chromosome der(21) (arrowed) which delimits the 11p region translocated on chromosome der(21); probes RP11‐691J8 (21q22.3) (B) and RP11‐1F8 (21q22.3) (C) spanning the breakpoint on 21, give a signal on both chromosomes 21 and on chromosome 21, but not on der(21) (arrowed), respectively. (D, E, F, G) Partial metaphases of patient 2. FISH with BAC probes located at 11p15.5. RP11‐3083I9 (D) and RP11‐1030I18 (E) show an increased hybridisation signal on chromosome dup(11) (arrowed) in comparison with that observed on normal chromosomes 11 (arrowhead): the two BACs target the most telomeric and centromeric duplicated regions in 11p15.5, respectively. CTD‐3176C5 (F) and CTD‐2242D18 (G) flanking the duplicated region telomerically and centromerically give a single hybridisation signal on chromosome dup(11) with a intensity and size comparable to that observed on normal chromosome 11. An additional signal on chromosome 12 (arrowhead) is observed by using probe CTD‐2242D18 (G). (H, I, L) Partial metaphases of patient 3. FISH with BAC RP11‐125N2 (11p15.4) (H) gives an additional hybridisation signal on der(4) (arrowed). Probe RP11‐120E20 (11p15.4) (I) gives a hybridisation signals on both chromosomes 11 delimiting the 11p duplicated region. Probe RP11‐452J12 (4p16.3) (L) shows the normal 4p hybridisation pattern, as it targets the non‐deleted most telomeric 4p region.

Figure 4 Physical map of breakpoint 11p clustering region. A physical map of the 11p15–p14 region containing the breakpoints (coloured arrows) underlying the rearrangements of the two BWS patients (patients 1 and 2) and the WHS patient (patient 3) is shown. The genomic clones used for FISH experiments are indicated by a black line. Black lines with a dot to the right indicate BAC clones that by FISH give hybridisation signals at multiple chromosomal locations. A few UCSC known genes are shown in blue. The grey/yellow/orange vertical lines or horizontal bars point to genomic regions detected as putative genomic duplications within the golden path (UCSC, May 2004). The vertical red bar indicates the presence of a gap clone. The scale refers to the May 2004 human draft sequence from the UCSC Genome Browser (see http://genome.ucsc.edu/cgi‐bin/hgGateway?org = Human).

Patient 2

FISH analysis with BAC clones mapping within the 11p15.5–p15.4 genomic interval (fig 4), confirmed the duplication of the 11p region (fig 3D,E) and defined its extent (1.8 Mb) by mapping of the telomeric and centromeric duplication breakpoints (fig 3F,G and online supplementary table 2). Interestingly, the centromeric breakpoint, falling between BAC RP11‐1030I18 and CTD2242D18, disrupts the amino‐terminal portion of the KCNQ1 gene.

Dual‐colour FISH using BACs localised in the duplicated region, demonstrated that the rearrangement is a direct duplication (see online supplementary fig 7 available at http://www.jmedgenet.com/supplemental).

Three colour FISH on the father's metaphases and nuclei using two BACs mapping within the duplicated interval, and one clone mapping outside, ruled out an inversion polymorphism of the duplication end points by showing BAC signals at the expected reciprocal positions (see online supplementary fig 8 available at http://www.jmedgenet.com/ supplemental).

Patient 3

The clinical features (table 1B) and family pedigree of patient 3 (fig 1C) suggested a translocation involving chromosome 4p segregating within the paternal line. FISH analysis performed with a commercial WHS probe detected a 4p16.3 deletion. Using subtelomeric probes, the unbalanced der(4)t(4;11)(p14.6;p15.4) translocation was indicated by the abnormal hybridisation pattern of 4p and 11p probes. Parental FISH analysis identified a balanced t(4;11) translocation in the father.

FISH analysis with BAC clones mapping within the 11p15.5–p15.4 and 4p16.3 genomic intervals (fig 4), delimited the 11p duplication (fig 3H,I) and the 4p deletion (fig 3L, table 2; online figs 6 and 9, both available at http://www.jmedgenet.com/supplemental).

BAC FISH characterisation established that the 11p duplication encompasses 3.4 Mb and the 4p deletion 4.6 Mb.

H19 and LIT1 methylation status in patient 2

Consistent with microsatellite and FISH results, proband 2 was heterozygous for HhaI RFLP, showing both paternal alleles after genomic digestion by the methylation sensitive HpaII endonuclease (fig 5A,B).

Figure 5 LIT1 and H19 methylation patterns in patient 2. (A) Pattern of segregation for HhaI RFLPs shows heterozygous status in father (F), mother (M), and proband 2 (P). (B) After HpaII methylation sensitive digestion, patient 2 (lane 2) shows two alleles corresponding to the two methylated paternal alleles, the maternal allele having been destroyed by HpaII digestion. Lanes 1 and 3 show a sample with only the uncut HhaI and cut HhaI sites, respectively. (C) Autoradiogram of LIT1 hybridisation shows the properly maternal methylated (6.0 kb fragment) and paternal unmethylated alleles (1.7 kb fragment) of patient 2 (lane 2) compared to a normal control (lane 1).

FISH evidence that the centromeric duplication breakpoint disrupts the KCNQ1 gene suggested that the methylation status of the paternally expressed transcript LIT1 might be altered. Methylation assay with a specific probe showed this was not the case as a proper methylation pattern with paternal unmethylated and maternal methylated alleles could be observed (fig 5C).

Discussion

As assessed by microsatellite analysis and FISH characterisation, both patients 1 and 3 carry an unbalanced translocation leading to paternal duplication of 11p15.4‐pter: the duplicated regions extend 3.8 and 3.4 Mb, respectively, and the breakpoint cluster is in a small 11p15.4 region, lying about 400 kb away, as shown in the physical map of fig 4 (green and blue arrows). Despite this striking similarity, monosomy affecting 3.5 Mb of telomeric 21q and 4.6 Mb of telomeric 4p partner chromosomes combines with overrepresentation of 11p15.4‐pter to yield a quite distinct phenotypic spectrum. Indeed a few signs are displayed by patient 1 which can be considered extra to the typical BWS spectrum and hence are imputed to monosomy of distal 21q, such as the slight developmental delay which is an uncommon sign of pure BWS syndrome. Interestingly, the predominant WHS phenotype exhibited by patient 3 adds further evidence to the prevalent phenotype of monosomy 4p, already reported in WHS cases arising from unbalanced segregation of a familial translocation, even when the duplicated region causes a well defined syndrome, as in BWS. To our knowledge, only one other WHS paternally derived familial translocation involving 11p15.5, not characterised by molecular cytogenetics, has been reported.²⁵ As regards BWS criteria, proband 1 and his deceased brother taken together fit the wide spectrum of the major criteria of the syndrome, including overgrowth/gigantism, omphalocele, and embryonal tumour, while proband 2, carrying a smaller 11p15 duplicated region, appears to have a less severe phenotype, with hemihypertrophy and macroglossia as major features, but shows a wider range of facial dysmorphisms, similar to the facial features of the recently described female WHS patient.²⁶ Strikingly, the two patients share the same full‐cheeked appearance of the face, the wide and depressed nasal bridge, and the presence of a large haemangioma. Moreover Stevenson's patient had neonatal persistent hypoglycaemia, accepted as a minor finding associated with BWS. As for the dysmorphic features, the small region of overlap (∼700 bp telomeric to the BWS imprinted region) appears to modify even the dominant WHS phenotype.

The three chromosomal rearrangements described here appear to involve particular genomic motifs which are known to mediate, or enhance, the occurrence of aberrant inter‐ and intrachromosomal events.²⁷,²⁸,²⁹ Namely, the t(4;11) translocation is clearly mediated by a well known family of low copy repeats, the olfactory genes family (ORF),³⁰,³¹,³² through non‐allelic homologous recombination,²⁹ as both breakpoints fall within the clusters positioned on 4p16.3 and 11p15.4, respectively.³⁰ The t(11;21) breakpoints do not map within known duplicons, but LCR (low copy repeats) involvement is suspected as the 11p15.4 breakpoint is close to OR duplicons and the 21q22.3 breakpoint falls within a clone gap (chr 21: 43505734–45507092). Close to this gap we identified by BLAT a region (chr 21:43242877–43244759), as yet unreported, targeted by RP11‐146O16, which shows 92–93% homology with some OR regions,³¹,³² which sequences might have mediated the interchromosomal rearrangement. Consistent with this view is the involvement of the same 21q22.3 band in a BWS patient reported to carry an unbalanced t(11;21)(p15.2;q22.3).¹⁹ The pronounced instability of this 21q region is also attested by the presence of an LCV (large scale copy number variation) (variation 0234) identified at the centromeric breakpoint flanking region.³³ Most of the BWS translocations and/or inversions described in the literature,¹⁹,³⁴,³⁵ except for a few cases,¹⁹,³⁶,³⁷ affect chromosomal bands where OR genes or pseudogenes are located.³⁰,³¹ This non‐random occurrence would thus follow the trend documented for other LCRs.²⁹,³⁸ LCRs at 11p15 might also favour the occurrence of mitotic recombination leading to mosaic 11p uniparental disomy.

Particular to the direct tandem duplication of proband 2 is the lack of homology of duplication endpoint regions, although FISH probes, flanking the tel and cen breakpoints, were found to give a hybridisation pattern particular to segmental duplication regions. Indeed probes RP11‐51l17, CTD‐224D18, and RP11‐38L8I give hybridisation signals on several other chromosomes (see online table 2 and online fig 10, available at http://www.jmedgenet.com/supplemental). These novel findings are strongly suggestive of segmentons within 11p15.5, not yet reported on the UCSC Genome Browser or the Human Segmental Duplication Database, a region close to which novel variants have been recently reported.³⁹ Independent confirmation might contribute to further dissect the genomics of the highly unstable 11p15.5 interval. The small size, only 1.8 Mb, makes the observed duplication one of the smallest described in BWS, although comparison is difficult with the few reported apparently similar duplications lacking a refined characterisation.¹⁹ Most particularly, the centromeric duplication breakpoint falls within the amino‐terminus of KvLQT1, separating the two BWS IC domains without apparently modifying the methylation pattern of the paternally expressed antisense transcript LIT1. Conversely, balanced translocations with breakpoints on the maternal KvLQT1 transcript reported in the literature, cause BWS probably through alteration of LIT1 imprinting as consequence of a positional effect.⁴⁰ This finding is consistent with the independent regulation of IC1 and IC2, a hypothesis supported by the recent report on epimutation of IC1, which does not change the methylation status of LIT1, in patients affected with Silver‐Russell syndrome.⁴¹ Future studies exploiting this subtle rearrangement, shown by microsatellite segregation analysis and H19 methylation status to occur at paternal meiosis I, might provide further evidence on the autonomous regulation of the two BWS imprinted gene clusters.⁴²

Acknowledgements

The authors thank the patients and their families for agreeing to the studies. We also acknowledge the YAC Screening Centre of Dibit‐San Raffaele Scientific Institute (Milan, Italy) for providing the RPC11/1 BAC clones, and Dr M Rocchi (University of Bari, Italy) for providing the RPC11/2‐5 BAC clones.

Electronic‐database information

Supplementary table 2 and figs 6–10 are available at http://www.jmedgenet.com/supplemental. Online Mendelian Inheritance in Man (OMIM) is at http://www.ncbi.nlm.nih.gov/Omim/ (MIM 130650 for BWS; MIM 194190 for WHS); the GenBank electronic database is at http:/www.ncbi.nlm.nih.gov/Genbank; the UCSC Human Genome Browser is at http://genome.cse.ucsc.edu (release May 2004) (for physical mapping); the Database of Genomic Variants is at http://projects.tcag.ca/variation/; the Human Genome Segmental Duplication Database is at http://projects.tcag.ca/humandup; and the Database of Human Olfactory Receptor genes is at http:// bioinformatics.weizmann.ac.il/HORDE/humanGenes.

Abbreviations

BWS - Beckwith‐Wiedemann syndrome

FISH - fluorescent in situ hybridisation

UPD - uniparental disomy

WHS - Wolf‐Hirschorn syndrome