MATERIALS AND METHODS

Cytogenetic investigations

Chromosome analysis was performed on the proband’s blood using standard high resolution techniques. Fluorescent in situ hybridisation (FISH) with the commercially available probe WSR (Vysis Inc, Downers Grove, IL) was performed on the proband’s metaphase spreads. Other FISH experiments were performed with bacterial artificial chromosome (BAC) and prokaryotic artificial chromosome (PAC) clones labelled with biotin-dUTP (Vector Laboratories, Burlingame, CA) using nick translation; the labelled probes were visualised with FITC-avidin (Vector Laboratories) and the chromosomes were counterstained with DAPI (Sigma, Milano, Italy); hybridisations were analysed with a Zeiss Axioplan epifluorescence microscope and images captured with a Power Gene FISH System (PSI, Newcastle Upon Tyne, UK).

DNA analysis

Microsatellite analysis was conducted on peripheral blood DNA extracted by standard techniques following the protocol described in Perez-Jurado et al.⁹ The portion of intron 1 of the CYLN2 gene containing the previously unreported 4 bp microsatellite polymorphism was amplified with primers CYLN2 i1F (5′-CTCTTCCCTTTCGGTTGTAAATGT-3′, ABI-Fam labelled) and CYLN2 i1R (5′- CGCCTCCACCTGCCTCTTCT-3′) and the same PCR protocol used for the other polymorphisms. All primers were purchased from MWG Biotech (Ebersberg, Germany). The 473/477 bp fragments were visualised like all other polymorphisms on an ABI PRISM 310 Genetic Analyzer (Applera, Monza, Italy). The CYLN2 i1 polymorphism was verified in 50 unrelated normal subjects: 36 were 473/473, 13 473/477, and one was 477/477. Allele frequencies were 0.85 for the 473 bp allele and 0.15 for the 477 bp allele. All sequencing reactions were performed with a Big Dye terminator cycle sequencing kit (Applera). Sequences were run on an ABI Prism 310 Genetic Analyzer.

RESULTS

Clinical description

The proband is a 5½12 year old boy, the second child of healthy, unrelated parents. Written informed consent for the neuropsychological examination and the genetic analysis was obtained from his parents. The pregnancy was uneventful, except for mild intrauterine growth retardation during the third trimester, shown by ultrasound examination. At birth the child’s weight was 2450 g (25th centile), his length was 45 cm (3rd centile), and his head circumference was 34 cm (25th centile). His Apgar scores were 10/10. He had supravalvular aortic stenosis (SVAS, surgically corrected at the age of 23 months) and vesicoureteric reflux with right megaureter (surgically corrected at the age of 17 months). The following facial dysmorphism was evident: mild coarsening of facial features, bitemporal narrowing, sparse eyebrows, downward slanting palpebral fissures, deep set eyes, prominent cheeks, bulbous triangular nasal tip, long and featureless philtrum, and macrostomia (fig 1).

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Figure 1

Photograph of the subject aged 5½12 years.

Neurological evaluation was normal, except for slightly decreased muscle tone, with hypertonia of the tibiotarsal joints and increased deep tendon reflexes. Balance was adequate.

Neuropsychological testing

The patient’s psychomotor milestones were mildly delayed; he sat at the age of 8 months, walked at 12 months, and spoke his first words at 18 months and first sentences at 36 months. His cognitive profile was assessed at the age of 5½12 years. We used a general intelligence test, the Stanford Binet Development Scale,¹⁹ and a neuropsychological battery, VMI block construction²⁰ and Rey Figure,²¹ in order to assess visual-spatial and visuoconstructive abilities; verbal and spatial working memory and recall memory for drawings were assessed respectively by Digit Span, Corsi Span,²² and Rey Figure Memory task (table 1). The subject showed borderline IQ (IQ=83), with some difficulties in vocabulary (mildly restricted), syntactic organisation (poor), and comprehension. His performance in visual-spatial and visuoconstructive tasks was borderline, like his IQ. Verbal and spatial working memory were mildly impaired, without significant differences between verbal and non-verbal domains. His social behaviour was friendly, and no anxiety trait or overfriendly manner could be detected.

The patient’s performance was compared to a group of age matched subjects (six boys and three girls, mean age 5.12 years, SD 0.66) with Williams syndrome and the typical deletion (Williams syndrome Control Group, WSCG), who underwent the same test battery (table 1). The WSCG’s performance was characterised by mild cognitive impairment (IQ=68.67, SD 16.29), major impairment in non-verbal abilities, mainly in visuoconstructive tasks, and a very low level of spatial organisation; verbal working memory was better than spatial. The WSCG displayed the typical Williams syndrome cognitive profile. The proband’s and WSCG’s test performances are compared in the last column of table 1. The difference in IQ was statistically significant. The patient’s performance was similar to the WSCG in language tasks, but significantly better in spatial and constructive organisation. In fact the nine children of the WSCG were not able to perform the Rey Figure task (copy and memory), which is sensitive to the ability to organise spatially and hierarchically a graphic performance by first copying from a model and later recalling it, while the proband performed at a level adequate to his mental age.

Similar data were obtained from the second copy and draw test, the VMI test. No significant differences with the WSCG were shown by the other tests (block construction, Digit and Corsi Span).

Genetic analysis

FISH analysis using the commercially available probe WSR (Vysis) showed hemizygosity at the ELN, LIMK1, and D7S613 loci. FISH with clone CTB-8H17, partially overlapping the centromeric cluster of repeated sequences²³ (BAC 1008H17), detected a partial deletion showing a small signal on one chromosome 7 compared to its homologue (fig 2A). The result was confirmed by performing a FISH analysis on a subject with typical WS deletion; the probe shows the same pattern as observed in our patient (fig 2B), as previously described.²³ Additional FISH experiments with clones RP4-665P5²⁴ (fig 2C), RP11-815K3, and CTB-139P11²⁵ gave signals of equal intensity on both chromosome 7 homologues, indicating absence of deletion.

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Figure 2

FISH analysis of metaphase chromosomes from the patient (A, C) and from a control subject with a typical WS deletion (B). In the patient (A), FISH with BAC clone CTB-8H17 generates a small signal on one chromosome 7 (arrowhead) compared to the other (arrow), indicating a partial deletion. The same result is obtained analysing a subject with a typical WS deletion (B). The clone CTB-8H17, partially overlapping the centromeric cluster of repeated sequences, gives a partial deletion pattern in subjects with the typical WS deletion, as described previously.²³ FISH with PAC probe RP5-665P05 (C) shows signals of equal intensity on both chromosomes 7 (arrows).

Microsatellite analysis⁹ on peripheral blood DNA from the patient, his brother, and his parents confirmed hemizygosity for marker D7S613 and the paternal origin of the deletion, but showed dizygosity for D7S1870. Markers D7S653, D7S1816, D7S489A, and D7S669 were also heterozygous. All other markers were uninformative (fig 3). The proband and his brother inherited different chromosome 7 haplotypes from their mother and the same haplotype from their father. In the proband the deletion was not associated with a recombination.

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Figure 3

Molecular genotyping of the proband and his family with microsatellite probes in the WS region. The numbers indicate allele size in base pairs. Alleles belonging to the same haplotype have been vertically aligned and shaded. In the proband, markers D7S489B and D7S2476 have been drawn as deleted based on the FISH results with probe CTB-8H17; marker D7S2472 has been drawn as non-deleted because of FISH results with probe RP5-665P05; marker CYLN2 i1 has been considered deleted for the reasons outlined in the results section.

Typing of a previously undescribed 4 bp (TTCA) insertion/deletion polymorphism in intron 1 of the CYLN2 gene (1066 bp downstream of exon 1; position 11736822 on sequence NT_007758.8) showed that the proband’s deletion includes at least the 5′ end of the gene. In fact, the proband and his brother inherited the same paternal haplotype carrying the 477 bp CYLN2 i1 allele, but the proband only has the 473 bp allele (fig 3). He could have inherited his father’s 473 bp allele only in the unlikely event of a double crossover. Sequence analysis of all RFC2¹² and CYLN2¹⁶ exons and of several intronic single nucleotide polymorphisms (SNPs) in the region (not shown) did not show any other informative polymorphism.

These results map the extent of the deletion from the typical WS breakpoint on the centromeric side to between intron 1 of CYLN2 and PAC clone RP4-665P5 on the telomeric side (fig 4).

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Figure 4

Physical map of the WS region at 7q11.23 (not drawn to scale) showing the relative location of the probes used for FISH analysis (thick horizontal lines), informative polymorphic markers (vertical lines), and selected genes (arrows).

DISCUSSION

The subject described here meets the diagnostic criteria for WS (clinical features, SVAS, short stature, positive FISH analysis), but his clinical and neuropsychological profiles are unusual. Face features are typical for WS, but more mildly expressed; moreover, inspection of photographs taken at different periods through infancy and childhood showed a gradual reduction of WS facial features (data not shown). The cognitive profile displayed by the patient was different in a number of ways from the WSCG. He had borderline IQ, while the age matched WSCG children were mildly impaired. He did not show the usual WS behavioural pattern. Moreover, his cognitive profile did not show the typical spatial and constructive impairment. However, the patient showed impairment in several competencies. In this sense, his development differs from both the WSCG and normally developing children.

Very few subjects with deletions that do not span the entire WS region have been reported so far.^{8,14,23,26–29} Two patients carrying a deletion spanning from ELN to marker D7S1870 still had the full WS phenotype.²⁶ Tassabehji et al¹⁴ and Karmiloff-Smith et al²⁸ accurately described a SVAS patient carrying a large deletion encompassing all genes in the WS region with the exception of CYLN2, GTF2IRD1, and GTF2I, but no clinical and cognitive WS phenotype. They used the British Abilities Scale II (BAS) scale for the assessment of cognitive abilities, and showed “an above average, even cognitive profile, with no indication of spatial impairment.” Since there is no standardisation of the BAS scale for the Italian population, we used a different cognitive assessment, but explored the same functional areas.

Our patient’s profile is different from that of the subject they described because of his borderline cognitive abilities but similar in the absence of the typical spatial constructive impairment. In the subject described by Korenberg et al²³ (subject RM1199), all genes between FZD9 and WSCR1 were deleted, while RFC2, CYLN2, GTF2IRD1, and GTF2I were not. This 8 year old girl had SVAS, some minor dysmorphisms, and mild mental retardation. No information on her cognitive or behavioural profile was available. Del Campo et al²⁹ reported a family with SVAS, borderline mental functioning, gregarious personality, minor facial WBS, and absence of visual/spatial deficits. Molecular analysis showed a 700 kb deletion including all genes from ELN to GTF2IRD1.

The GTF2I gene, located in the telomeric copy of the WS typical breakpoint region and deleted in all WS subjects with a typical deletion,³⁰ is not deleted in our patient. This gene encodes BAP-135, a protein phosphorylated by Bruton’s tyrosine kinase, as well as the transcription factor TFII-I. The centromeric copy of the WS breakpoint region contains a highly similar (99.9% throughout the coding region) transcribed pseudogene, GTF2IP1.³⁰ The GTF2IRD1 gene, coding for a putative transcription factor with ubiquitous expression,^31,32 may also be preserved. All the other genes in the WS region are deleted.

We cannot rule out that in our patient CYLN2 may be expressed from an alternative promoter located downstream from the known exon 1, since the transcription pattern of human CYLN2 has not been fully analysed and its protein coding region starts in exon 2.¹⁶ On the other hand, a deletion involving at least exon 1 and the entire upstream regulatory portion of the gene is likely to have major effects on its expression.

Tassabehji et al¹⁴ suggested that all determinants of the WS phenotype, apart from SVAS, lie telomeric to RFC2. Our observations indicate that, while the WS phenotype is the result of the haploinsufficiency of a number of genes, the deletion of the GTF2IRD1 and/or GTF2I genes located on the telomeric side of the WS region is necessary for the syndrome’s unique cognitive profile. Transgenic mice carrying a disrupted Gtf2ird1 gene³³ did not show any obvious impairment, although it must be pointed out that they were not tested in detail for subtle cognitive and behavioural defects. It is also interesting to note that Osborne et al²⁴ described a subject with WS facies, developmental delay, and WS-like behavioural profile (subject 12503) carrying an inversion of the WS region, and hypothesised that GTF2I may have been affected by the rearrangement. The GTF2I gene may then be critical for the WSCP.

Our data also show that hemizygosity for LIMK1 and CYLN2, while not sufficient to generate the WSCP, may cause alterations in the cognitive profile. Very likely, deletion of GTF2I, GTF2IRD1, and CYLN2 (and perhaps LIMK1) is necessary to cause the typical WSCP. When, as in the case of our patient, one or more of these genes is preserved, the result is a milder phenotype, with some cognitive impairment (borderline IQ) and a variable loss of visual-spatial and constructive abilities.

Identification of additional subjects with atypical deletions, careful comparison of their genetic, clinical, and neuropsychological profiles, and the development of methods for the molecular analysis of GTF2I will be needed in order to assess the contribution of each gene to the WS phenotype.