Severe Expressive-Language Delay Related to Duplication of the Williams–Beuren Locus
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《新英格兰医药杂志》
SUMMARY
The Williams–Beuren syndrome (WBS) locus, at 7q11.23, is prone to recurrent chromosomal rearrangements, including the microdeletion that causes WBS, a multisystem condition with characteristic cardiovascular, cognitive, and behavioral features. It is hypothesized that reciprocal duplications of the WBS interval should also occur, and here we present such a case description. The most striking phenotype was a severe delay in expressive speech, in contrast to the normal articulation and fluent expressive language observed in persons with WBS. Our results suggest that specific genes at 7q11.23 are exquisitely sensitive to dosage alterations that can influence human language and visuospatial capabilities.
The underlying genetic bases for the majority of cases of language impairment have been postulated to be complex, involving several loci that interact with one another and the environment to produce an overall susceptibility to disease onset.1 Clues to the discovery of which genes potentially influence language ability may be found in mendelian disorders that have distinctive language components to their clinical phenotype. The Williams–Beuren syndrome (WBS) is one such neurodevelopmental disorder, in which persons show considerable strength in expressive language relative to their overall level of intellectual ability.2 WBS is also associated with a recognizable facies, supravalvular aortic stenosis, hypersensitivity to sound, visual impairment, dental problems, growth deficiency, infantile hypercalcemia, musculoskeletal abnormalities, and a hoarse voice.3 The syndrome is caused by the recurrent deletion of a specific set of genes, so it provides a unique opportunity to identify genes that are directly involved in language ability.4
The chromosomal locus that is deleted in WBS (on chromosome 7, band q11.23) is prone to deletion because it is flanked by blocks of DNA that have a very high degree of similarity to one another (called low copy repeats ).4 The deletions, which almost invariably span a common interval, are caused by nonallelic homologous recombination within the LCRs of either the same chromosome 7 (i.e., intrachromosomal) or different chromosome 7s (i.e., interchromosomal). In each case, the chromosomes are envisaged to form loops, thereby allowing the alignment of the two LCRs, the occurrence of recombination, and the excision of the DNA contained within the intervening loop.4 The syndrome occurs at a frequency of approximately 1 in 7500 live births, with approximately two thirds of the deletion events being interchromosomal.5 Other microdeletion disorders — including the velocardiofacial syndrome, the Smith–Magenis syndrome, the Prader–Willi and Angelman syndromes, and hereditary neuropathy with liability to pressure palsies — are also mediated by nonallelic homologous recombination.6 For each of these microdeletions, a reciprocal duplication disorder has also been identified: dup22q11.2, dup17p11.2, dup15q11-q13, and Charcot–Marie–Tooth type 1A, respectively.6
Theoretically, duplications of the WBS region should occur at the same frequency as deletions occurring through interchromosomal nonallelic homologous recombination6 (i.e., at a frequency of 1 in 13,500). However, such duplication has not yet been described, even though there has been ample opportunity for the observation of such rearrangements in studies of patients with WBS-like clinical features but no deletion.4,7,8 That duplications have not been found in these patients suggests that the effect of duplication of the WBS locus is either lethal or has no observable consequence. Alternatively, the duplication could result in a clinical presentation that does not overlap with that of WBS and is therefore not represented in this study population. The patient we describe here, who has an exact duplication of the WBS region, has a phenotype that includes a severe delay in expressive language — a characteristic that is distinct from any of the typical clinical features seen in WBS.
Case Report
The proband (Patient 1) was born after a full-term pregnancy complicated by maternal Crohn's disease and poor weight gain. (Patient 1's mother died when he was four years three months old.) His birth weight was 2520 g (below the 5th percentile), his length was 44.5 cm (below the 5th percentile), and his head circumference was 33 cm (at the 10th percentile). He initially required gavage feeding and was evaluated for failure to thrive and hypotonia at 13 months. According to a parental report, he sat at one year and walked at two years. He was evaluated by a speech pathologist and given diagnoses of moderate-to-severe language delay at the age of two years and severe delay in receptive and expressive language at the age of three years two months. When he was four years two months of age, attention deficit–hyperactivity disorder (ADHD), an overall developmental delay, an unspecified sleep disorder, and a severe delay in speech and language and in fine motor skills were diagnosed. Results of behavioral audiometry and otoacoustic emissions were consistent with the presence of normal hearing. At the age of eight years, Patient 1 was again evaluated by a speech pathologist, and childhood apraxia of speech was diagnosed. Additional clinical, biochemical, and genetic analyses did not reveal any abnormalities (see the Supplementary Appendix, available with the full text of this article at www.nejm.org).
Patient 1's sister (Patient 2) was also given a diagnosis of ADHD, and both were receiving 5 mg of dextroamphetamine sulfate (Dexedrine) twice daily. The history revealed that both parents had also had attention and academic difficulties but that Patient 1 was the only member of the family who had difficulty with language acquisition.
Written informed consent was obtained from the guardians of the patients for genetic and psychological studies, which were approved by institutional ethics review boards. At the time of our assessment, Patient 1 was 8 years 10 months old, and Patient 2 was 11 years 1 month old.
Mild Physical Manifestations
Patient 1 had growth retardation and mild dysmorphism. His physical examination showed height, weight, and head circumference at the 2nd, 5th, and 30th percentiles, respectively. Dysmorphic features included dolichocephaly, a high and narrow forehead, long eyelashes, a high and broad nose, a short philtrum, a high-arched palate, dental malocclusion (specifically, an anterior open bite), retrognathia, and asymmetric crying face (Figure 1 and Table 1). Patient 1 did not have any of the facial features of WBS.3 He had bilateral simian creases, and the left hand was smaller than the right. On neurologic examination, he was noted to have very mild dysmetria and mild difficulty with tandem gait and unipedal stance.
Figure 1. Photographs of Patient 1 at the Age of Eight Years.
Patient 1 has a high and narrow forehead, a high and broad nose, a short philtrum, and facial asymmetry (Panel A). In Panel B, long eyelashes and retrognathia can be clearly seen.
Table 1. Clinical and Neurobehavioral Differences between Williams–Beuren Syndrome (WBS) and WBS Region Duplication.
Severe Delay in Expressive Language
Patient 1's difficulties with expressive language were immediately apparent. He was able to pronounce only a very small number of words correctly; most words were approximations composed of the first consonant (or a related consonant) and the first vowel (or a related or neutral vowel) of the target word. The results of the intellectual and vocabulary assessments are summarized in Table 2. Patient 1's performance on the Differential Ability Scales9 differed from his sister's only on the verbal cluster, in which verbal responses, manual signs, gestures, pantomime, and drawing were all considered acceptable responses from him. If only verbal responses had been accepted, his verbal-cluster standard score would have been considerably lower.
Table 2. Standard Scores on Intellectual and Vocabulary Assessments.
To provide a direct comparison of Patient 1's receptive and expressive language abilities, the Peabody Picture Vocabulary Test,10 which measures receptive vocabulary, and the Expressive Vocabulary Test11 were administered (Table 2). Patient 1's standard score on receptive vocabulary was in the low-average range (age equivalent, 6 years 10 months). In contrast, his standard score on expressive vocabulary was in the severe-impairment range (age equivalent, two years three months), even though both word approximations and manual signs were considered acceptable responses. Similar results were obtained with the Clinical Evaluation of Language Fundamentals test12 (see the Supplementary Appendix).
Although Patient 1's standard scores on the nonverbal reasoning and spatial clusters of the Differential Ability Scales were in the range of mild mental deficiency, his performance was very similar to his sister's and was consistent with the type of difficulties his mother was also reported to have had, indicating that the duplication was not the primary basis for these difficulties. Both children's scores on the Conners' scales13 (found in the Diagnostic and Statistical Manual of Mental Disorders, 4th ed.) were consistent with their previous diagnosis of ADHD.
Methods and Results
Duplication of the 1.5-MB WBS Region
Patient 1 was initially referred for testing for the velocardiofacial syndrome and was screened with the use of a real-time method on the basis of a polymerase-chain-reaction (PCR) assay, which detected the duplication of markers within the WBS critical region.14 Fluorescence in situ hybridization (FISH) with probes from 7q11.237 showed that the in-tandem duplication was limited to the region commonly deleted in WBS (Figure 2A). Probes from within the WBS common deletion (CTA-208H19 and RP5-1186P10) gave three signals on interphase FISH, whereas probes flanking the deletion region (RP11-815K3 and CTB-139P11) gave only two signals (Figure 2A; some data not shown). Cosmid LL07NCO1-207g3, which lies between the medial and telomeric LCRs, also gave only two signals, indicating that the duplication was restricted to the region spanning the centromeric and medial LCRs, which corresponds to the region commonly deleted in WBS. No duplication was present in Patient 1's father or sister or in more than 250 other controls with or without WBS who were also examined.
Figure 2. Schematic Representation of the 7q11.23 Duplication.
In Panel A, analysis by fluorescence in situ hybridization (FISH) was performed on interphase and metaphase spreads prepared from both fresh lymphocytes and lymphoblastoid cell lines, as described previously,7 and in all cases the results were the same. Shown is FISH analysis of Patient 1 with clones from within the region (CTA-208H19 and RP5-1186P10) that is commonly deleted in the Williams–Beuren syndrome (WBS) and from an area flanking that region (CTB-139P11 and cos207g3). Fluorescent signals are grouped into those from the normal chromosome 7 (N Chr 7) and from the duplicated chromosome 7 (Dup Chr 7). A schematic representation of the location of the probes on each chromosome is also shown. The chromosomes are oriented with the centromere at the top, and the low copy repeats (LCRs) are depicted as gray boxes. The probes are depicted in colors corresponding to the FISH image. The 1.5-Mb region of chromosome 7q11.23 that is commonly deleted in WBS is indicated. Panel B shows a map of the region of exchange in block B leading to duplication of the common 1.5-Mb WBS deletion region. Polymorphic microsatellite markers and genes from the WBS region are shown on the top. The unique sequence is depicted by gray lines, and the repetitive blocks that constitute the LCRs are depicted as black arrows, indicating their orientation on the chromosome. A detailed schematic representation of the entire 143-kb length of block B is also shown. The dotted arrow on the right shows that the last 38 kb is absent in the centromeric B block (Bc). The genomic structure of genes within the region is also shown. The genes are those encoding general transcription factor 2 I (GTF2I), neutrophil cytosolic factor 1 (NCF1), and GTF2I repeat domain containing protein 2 (GTF2IRD2). Functional copies of these genes are present in block Bm, and corresponding pseudogenes (P1 or P2) are present in blocks Bc and Bt, except for the Bt copy of GTF2IRD2 that appears to be a functional gene as well. Exons are depicted as black boxes, and the numbers indicate the first and last exons within the block. Numerical identification, location, and sequence difference of each site-specific nucleotide (SSN) that was used in this study are indicated and have been described previously.4 All dosage-analysis calculations were performed with regard to Patient 1, who carried one normal chromosome 7, and his father, who carried two normal chromosomes 7. The inferred recombinant block B in the duplicated chromosome is shown at the bottom. The predicted genotypes at each position are depicted as circles: white circles denote Bc-type, black circles Bm-type; and black-and-white circles either Bc-type or Bm-type. The region of exchange occurred somewhere between SSN 4 and SSN 6 within the NCF1 gene and one of its pseudogenes.
Analysis of single-copy microsatellite markers from within the WBS region revealed that Patient 1 carried three distinct alleles at loci D7S2476, D7S3194, and D7S1870 (Table 1 of the Supplementary Appendix). In each case, only one of the alleles was present in the father, indicating that Patient 1 had inherited two different copies of the WBS region from his mother. Analysis of the maternal grandparents revealed that the duplicated chromosome in the proband contained segments of chromosome 7 that were inherited independently from each maternal grandparent (Table 1 and Figure 1 of the Supplementary Appendix). These analyses indicate that the meiotic interchromosomal recombination that led to the duplication took place in the mother's germ cells, making it a new rearrangement.
Reciprocal of the WBS Deletion
Further narrowing of the duplication breakpoints could not be accomplished with FISH or microsatellite analysis, owing to the high sequence identity of the LCRs. We used site-specific nucleotide (SSN) dosage analysis to define the relative number of B block–type copies (centromeric, medial, or telomeric) at each position analyzed, an approach that enabled us to infer the position where the recombination had occurred.4 Analysis of seven SSNs showed that in Patient 1 the transition between blocks Bm and Bc occurred between SSN 4 and SSN 6, within the NCF1 gene (Figure 2B). The majority of WBS deletion breakpoints (>90 percent) also occur within the B block,4 indicating that the duplication is the exact reciprocal of the common WBS deletion.
Altered Expression of Genes within the Duplication
Gene-expression analysis in lymphoblastoid cell lines by real-time PCR demonstrated that five of six genes that were examined within the duplicated region (GTF2I, LIMK1, WBSCR1, RFC2, and BAZ1B) showed increased expression in Patient 1 and reduced expression in persons with WBS (Table 2 of the Supplementary Appendix). The exception was WBSCR5, which showed reduced expression in persons with WBS but levels in Patient 1 that were consistent with levels in controls. WBSCR16, located just outside the telomeric deletion or duplication, did not show altered expression in either Patient 1 or in persons with WBS, indicating that the rearrangement breakpoints do not affect its transcription. The nearest gene outside the centromeric breakpoint, the gene encoding calneuron 1, was not expressed in lymphoblasts, but it is at least 300 kb from the proximal B block4 and is separated from the breakpoint by the complex and actively transcribed LCRs; hence, its expression unlikely to be affected.
Discussion
We describe a syndrome that is associated with a reciprocal duplication of the WBS microdeletion region. Patient 1 has intellectual strengths and weaknesses that are in direct contrast to those of children with WBS. Expressive language, especially syntax and phonology, is the area of greatest weakness for Patient 1, whereas for children with WBS, expressive language is a relative strength.2 When Patient 1 had difficulty making himself understood, he often successfully resorted to drawing what he was trying to express. Visuospatial construction (including drawing) is the area of greatest weakness for children with WBS, and most children with WBS who are Patient 1's age are able to draw only a few recognizable objects.15
Previous descriptions of persons with larger duplications of the region (supernumerary ring chromosome 7) have noted delay in expressive language or impairment accompanied by articulation problems, but none of these reports contain standardized assessment results or comparative data on expressive and receptive language.16 Karyotypes were derived from G-banding, not from molecular analysis, so the extent of each duplication remains unknown. Our findings specify the expressive-language phenotype associated with dup7q11.23 and define the precise region of chromosome 7 contributing to it as the 1.5-Mb interval commonly deleted in WBS.
There appears to be a subtle but recognizable facial phenotype that is shared by both Patient 1 and previously described persons with supernumerary ring chromosome 7, one that consists of a high and broad nose, posteriorly rotated ears, a high-arched palate, and a short philtrum.16,17,18 When accompanied by delay in expressive language, this gestalt warrants testing for duplication of the WBS critical region.
Although there is a strong genetic component to language impairment, so far only the transcription factor FOXP2 has been implicated as a cause of the problem, and only in a few cases.19 Disruption of FOXP2 results in reduced functional dosage and leads to deficits in both expressive and receptive language, in addition to orofacial dyspraxia that impairs the coordination of complex fine-motor movements of the lower face.19,20 The remaining cases of language impairment were predicted to involve complex causes.1 The identification of a second locus associated with language impairment is therefore unexpected and opens up the possibility of linking the expressive-language phenotype to a specific gene or genes from within the WBS region.
The contrast between phenotypes associated with deletion and those associated with duplication of the WBS region, as well as the accompanying changes in gene expression, suggests that genes within this region are dosage sensitive. These observations also suggests that perturbation can have a dramatic, negative effect on both language development and visuospatial construction ability.
The duplication or deletion encompasses 26 to 27 genes, many of which can be ruled out as important contributors to the WBS phenotype on the basis of the correlation of genotype with phenotype in persons with atypical deletions.21,22,23,24 The minimal critical interval that must be deleted in classic WBS spans the region between the gene encoding elastin and the common distal breakpoint and encompasses just nine genes. Included within the WBS minimal critical interval are three general transcription factor 2 I (GTF2I) genes, encoding a family of proteins that are predicted to possess some functional redundancy.25 In a simple or more complex model, alteration of expression of a single gene — or in the case of the GTF2I family, possibly a combination of genes — might lead to a distinctive language-impairment phenotype that overlaps with that observed in Patient 1. Our discovery of the WBS duplication provides both the long-sought-after reference case for ascertainment of other patients and an entry point toward the discovery of new genes required for the normal acquisition and expression of language. The association between expressive-language delay and duplication of the WBS region also has implications for the molecular diagnosis of language delay.
Supported by grants from the Canadian Institutes of Health Research and the Sick Kids Foundation (both to Dr. Osborne), the Spanish Ministries of Health and Science and Technology (to Dr. Pérez-Jurado), Genome Canada/Ontario Genomics Institute and Genome Spain (to Drs. Pérez-Jurado, Scherer, and Osborne), the National Institute of Neurological Disorders and Stroke (NS35102, to Drs. Mervis and Morris), and the National Institute of Child Health and Human Development (HD29957, to Dr. Mervis). Dr. Scherer is a Canadian Institutes of Health Research investigator and an international scholar of the Howard Hughes Medical Institute, and Dr. Osborne is a Canadian Institutes of Health Research scholar.
We are indebted to the patient and his family for their participation in our study and to the Centre for Applied Genomics at the Hospital for Sick Children, Toronto, for technical assistance.
Source Information
From the Department of Medical Genetics, University of Alberta, Edmonton, Alta., Canada (M.J.S., S.B., M.L.); the Department of Psychological and Brain Sciences, University of Louisville, Louisville, Ky. (C.B.M., E.P.); the Institute of Medical Science (E.J.Y., L.R.O.) and the Departments of Molecular and Medical Genetics (W.L., S.W.S., L.R.O.) and Medicine (L.R.O.), University of Toronto, and the Program in Genetics and Genomic Biology, Hospital for Sick Children (E.-J.S., S.W.S.) — both in Toronto; the Unitat de Genètica, Departament Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona (M.C., L.A.P.-J.); and the Department of Pediatrics, University of Nevada School of Medicine, Las Vegas (C.A.M.).
Drs. Somerville and Mervis contributed equally to this article.
Address reprint requests to Dr. Osborne at the Department of Medicine, University of Toronto, 7238 Medical Sciences Bldg., 1 King's College Circle, Toronto, ON M5S 1A8, Canada, or at lucy.osborne@utoronto.ca.
References
Fisher SE, Lai CS, Monaco AP. Deciphering the genetic basis of speech and language disorders. Annu Rev Neurosci 2003;26:57-80.
Mervis CB, Klein-Tasman BP. Williams syndrome: cognition, personality, and adaptive behavior. Ment Retard Dev Disabil Res Rev 2000;6:148-158.
Morris CA. Williams syndrome. In: Cassidy SB, Allanson JE, eds. Management of genetic syndromes. 2nd ed. Hoboken, N.J.: John Wiley, 2005:655-65.
Bayés M, Magano LF, Rivera N, Flores R, Pérez Jurado LA. Mutational mechanisms of Williams-Beuren syndrome deletions. Am J Hum Genet 2003;73:131-151.
Stromme P, Bjornstad PG, Ramstad K. Prevalence estimation of Williams syndrome. J Child Neurol 2002;17:269-271.
Inoue K, Lupski JR. Molecular mechanisms for genomic disorders. Annu Rev Genomics Hum Genet 2002;3:199-242.
Osborne LR, Li M, Pober B, et al. A 1.5 million-base pair inversion polymorphism in families with Williams-Beuren syndrome. Nat Genet 2001;29:321-325.
Scherer SW, Cheung J, MacDonald JR, et al. Human chromosome 7: DNA sequence and biology. Science 2003;300:767-772.
Elliott CD. Differential ability scales: introductory and technical handbook. San Antonio, Tex.: Psychological Corporation, 1990.
Dunn LM, Dunn LM. Peabody picture vocabulary test. 3rd ed. Circle Pines, Minn.: American Guidance Service, 1997.
Williams KT. Expressive vocabulary test. Circle Pines, Minn.: American Guidance Service, 1997.
Wiig EH, Secord WA, Semel E. Clinical evaluation of language fundamentals preschool. 2nd ed. San Antonio, Tex.: Psychological Corporation, 2004.
Conners CK. Conners' rating scales — revised. Toronto: Multi-Health Systems, 1997.
Christiansen J, Dyck JD, Elyas BG, et al. Chromosome 1q21.1 contiguous gene deletion is associated with congenital heart disease. Circ Res 2004;94:1429-1435.
Bertrand J, Mervis CB, Eisenberg JD. Drawing by children with Williams syndrome: a developmental perspective. Dev Neuropsychol 1997;13:41-67.
Lichtenbelt KD, Hochstenbach R, van Dam WM, Eleveld MJ, Poot M, Beemer FA. Supernumerary ring chromosome 7 mosaicism: case report, investigation of the gene content, and delineation of the phenotype. Am J Med Genet A 2005;132:93-100.
Tan-Sindhunata G, Castedo S, Leegte B, et al. Molecular cytogenetic characterization of a small, familial supernumerary ring chromosome 7 associated with mental retardation and an abnormal phenotype. Am J Med Genet 2000;92:147-152.
Chantot-Bastaraud S, Muti C, Pipiras E, et al. Clinical findings and cytogenetic analysis of small supernumerary ring chromosomes 7: report of two new cases. Ann Genet 2004;47:241-249.
Lai CS, Fisher SE, Hurst JA, Vargha-Khadem F, Monaco AP. A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 2001;413:519-523.
Macdermot KD, Bonora E, Sykes N, et al. Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits. Am J Hum Genet 2005;76:1074-1080.
Tassabehji M. Williams-Beuren syndrome: a challenge for genotype-phenotype correlations. Hum Mol Genet 2003;Special No. 2:R229-R237.
Frangiskakis JM, Ewart AK, Morris CA, et al. LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell 1996;86:59-69.
Del Campo M, Magano LF, Martinez Iglesias J, Perez Jurado LA. Partial features of Williams-Beuren syndrome in a family with a novel 700 kb 7q11.23 deletion. Eur J Hum Genet 2001;9:Suppl 1:88-88. abstract.
Morris CA, Mervis CB, Hobart HH, et al. GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype-phenotype analysis of five families with deletions in the Williams syndrome region. Am J Med Genet A 2003;123:45-59.
Hinsley TA, Cunliffe P, Tipney HJ, Brass A, Tassabehji M. Comparison of TFII-I gene family members deleted in Williams-Beuren syndrome. Protein Sci 2004;13:2588-2599.(Martin J. Somerville, Ph.)
The Williams–Beuren syndrome (WBS) locus, at 7q11.23, is prone to recurrent chromosomal rearrangements, including the microdeletion that causes WBS, a multisystem condition with characteristic cardiovascular, cognitive, and behavioral features. It is hypothesized that reciprocal duplications of the WBS interval should also occur, and here we present such a case description. The most striking phenotype was a severe delay in expressive speech, in contrast to the normal articulation and fluent expressive language observed in persons with WBS. Our results suggest that specific genes at 7q11.23 are exquisitely sensitive to dosage alterations that can influence human language and visuospatial capabilities.
The underlying genetic bases for the majority of cases of language impairment have been postulated to be complex, involving several loci that interact with one another and the environment to produce an overall susceptibility to disease onset.1 Clues to the discovery of which genes potentially influence language ability may be found in mendelian disorders that have distinctive language components to their clinical phenotype. The Williams–Beuren syndrome (WBS) is one such neurodevelopmental disorder, in which persons show considerable strength in expressive language relative to their overall level of intellectual ability.2 WBS is also associated with a recognizable facies, supravalvular aortic stenosis, hypersensitivity to sound, visual impairment, dental problems, growth deficiency, infantile hypercalcemia, musculoskeletal abnormalities, and a hoarse voice.3 The syndrome is caused by the recurrent deletion of a specific set of genes, so it provides a unique opportunity to identify genes that are directly involved in language ability.4
The chromosomal locus that is deleted in WBS (on chromosome 7, band q11.23) is prone to deletion because it is flanked by blocks of DNA that have a very high degree of similarity to one another (called low copy repeats ).4 The deletions, which almost invariably span a common interval, are caused by nonallelic homologous recombination within the LCRs of either the same chromosome 7 (i.e., intrachromosomal) or different chromosome 7s (i.e., interchromosomal). In each case, the chromosomes are envisaged to form loops, thereby allowing the alignment of the two LCRs, the occurrence of recombination, and the excision of the DNA contained within the intervening loop.4 The syndrome occurs at a frequency of approximately 1 in 7500 live births, with approximately two thirds of the deletion events being interchromosomal.5 Other microdeletion disorders — including the velocardiofacial syndrome, the Smith–Magenis syndrome, the Prader–Willi and Angelman syndromes, and hereditary neuropathy with liability to pressure palsies — are also mediated by nonallelic homologous recombination.6 For each of these microdeletions, a reciprocal duplication disorder has also been identified: dup22q11.2, dup17p11.2, dup15q11-q13, and Charcot–Marie–Tooth type 1A, respectively.6
Theoretically, duplications of the WBS region should occur at the same frequency as deletions occurring through interchromosomal nonallelic homologous recombination6 (i.e., at a frequency of 1 in 13,500). However, such duplication has not yet been described, even though there has been ample opportunity for the observation of such rearrangements in studies of patients with WBS-like clinical features but no deletion.4,7,8 That duplications have not been found in these patients suggests that the effect of duplication of the WBS locus is either lethal or has no observable consequence. Alternatively, the duplication could result in a clinical presentation that does not overlap with that of WBS and is therefore not represented in this study population. The patient we describe here, who has an exact duplication of the WBS region, has a phenotype that includes a severe delay in expressive language — a characteristic that is distinct from any of the typical clinical features seen in WBS.
Case Report
The proband (Patient 1) was born after a full-term pregnancy complicated by maternal Crohn's disease and poor weight gain. (Patient 1's mother died when he was four years three months old.) His birth weight was 2520 g (below the 5th percentile), his length was 44.5 cm (below the 5th percentile), and his head circumference was 33 cm (at the 10th percentile). He initially required gavage feeding and was evaluated for failure to thrive and hypotonia at 13 months. According to a parental report, he sat at one year and walked at two years. He was evaluated by a speech pathologist and given diagnoses of moderate-to-severe language delay at the age of two years and severe delay in receptive and expressive language at the age of three years two months. When he was four years two months of age, attention deficit–hyperactivity disorder (ADHD), an overall developmental delay, an unspecified sleep disorder, and a severe delay in speech and language and in fine motor skills were diagnosed. Results of behavioral audiometry and otoacoustic emissions were consistent with the presence of normal hearing. At the age of eight years, Patient 1 was again evaluated by a speech pathologist, and childhood apraxia of speech was diagnosed. Additional clinical, biochemical, and genetic analyses did not reveal any abnormalities (see the Supplementary Appendix, available with the full text of this article at www.nejm.org).
Patient 1's sister (Patient 2) was also given a diagnosis of ADHD, and both were receiving 5 mg of dextroamphetamine sulfate (Dexedrine) twice daily. The history revealed that both parents had also had attention and academic difficulties but that Patient 1 was the only member of the family who had difficulty with language acquisition.
Written informed consent was obtained from the guardians of the patients for genetic and psychological studies, which were approved by institutional ethics review boards. At the time of our assessment, Patient 1 was 8 years 10 months old, and Patient 2 was 11 years 1 month old.
Mild Physical Manifestations
Patient 1 had growth retardation and mild dysmorphism. His physical examination showed height, weight, and head circumference at the 2nd, 5th, and 30th percentiles, respectively. Dysmorphic features included dolichocephaly, a high and narrow forehead, long eyelashes, a high and broad nose, a short philtrum, a high-arched palate, dental malocclusion (specifically, an anterior open bite), retrognathia, and asymmetric crying face (Figure 1 and Table 1). Patient 1 did not have any of the facial features of WBS.3 He had bilateral simian creases, and the left hand was smaller than the right. On neurologic examination, he was noted to have very mild dysmetria and mild difficulty with tandem gait and unipedal stance.
Figure 1. Photographs of Patient 1 at the Age of Eight Years.
Patient 1 has a high and narrow forehead, a high and broad nose, a short philtrum, and facial asymmetry (Panel A). In Panel B, long eyelashes and retrognathia can be clearly seen.
Table 1. Clinical and Neurobehavioral Differences between Williams–Beuren Syndrome (WBS) and WBS Region Duplication.
Severe Delay in Expressive Language
Patient 1's difficulties with expressive language were immediately apparent. He was able to pronounce only a very small number of words correctly; most words were approximations composed of the first consonant (or a related consonant) and the first vowel (or a related or neutral vowel) of the target word. The results of the intellectual and vocabulary assessments are summarized in Table 2. Patient 1's performance on the Differential Ability Scales9 differed from his sister's only on the verbal cluster, in which verbal responses, manual signs, gestures, pantomime, and drawing were all considered acceptable responses from him. If only verbal responses had been accepted, his verbal-cluster standard score would have been considerably lower.
Table 2. Standard Scores on Intellectual and Vocabulary Assessments.
To provide a direct comparison of Patient 1's receptive and expressive language abilities, the Peabody Picture Vocabulary Test,10 which measures receptive vocabulary, and the Expressive Vocabulary Test11 were administered (Table 2). Patient 1's standard score on receptive vocabulary was in the low-average range (age equivalent, 6 years 10 months). In contrast, his standard score on expressive vocabulary was in the severe-impairment range (age equivalent, two years three months), even though both word approximations and manual signs were considered acceptable responses. Similar results were obtained with the Clinical Evaluation of Language Fundamentals test12 (see the Supplementary Appendix).
Although Patient 1's standard scores on the nonverbal reasoning and spatial clusters of the Differential Ability Scales were in the range of mild mental deficiency, his performance was very similar to his sister's and was consistent with the type of difficulties his mother was also reported to have had, indicating that the duplication was not the primary basis for these difficulties. Both children's scores on the Conners' scales13 (found in the Diagnostic and Statistical Manual of Mental Disorders, 4th ed.) were consistent with their previous diagnosis of ADHD.
Methods and Results
Duplication of the 1.5-MB WBS Region
Patient 1 was initially referred for testing for the velocardiofacial syndrome and was screened with the use of a real-time method on the basis of a polymerase-chain-reaction (PCR) assay, which detected the duplication of markers within the WBS critical region.14 Fluorescence in situ hybridization (FISH) with probes from 7q11.237 showed that the in-tandem duplication was limited to the region commonly deleted in WBS (Figure 2A). Probes from within the WBS common deletion (CTA-208H19 and RP5-1186P10) gave three signals on interphase FISH, whereas probes flanking the deletion region (RP11-815K3 and CTB-139P11) gave only two signals (Figure 2A; some data not shown). Cosmid LL07NCO1-207g3, which lies between the medial and telomeric LCRs, also gave only two signals, indicating that the duplication was restricted to the region spanning the centromeric and medial LCRs, which corresponds to the region commonly deleted in WBS. No duplication was present in Patient 1's father or sister or in more than 250 other controls with or without WBS who were also examined.
Figure 2. Schematic Representation of the 7q11.23 Duplication.
In Panel A, analysis by fluorescence in situ hybridization (FISH) was performed on interphase and metaphase spreads prepared from both fresh lymphocytes and lymphoblastoid cell lines, as described previously,7 and in all cases the results were the same. Shown is FISH analysis of Patient 1 with clones from within the region (CTA-208H19 and RP5-1186P10) that is commonly deleted in the Williams–Beuren syndrome (WBS) and from an area flanking that region (CTB-139P11 and cos207g3). Fluorescent signals are grouped into those from the normal chromosome 7 (N Chr 7) and from the duplicated chromosome 7 (Dup Chr 7). A schematic representation of the location of the probes on each chromosome is also shown. The chromosomes are oriented with the centromere at the top, and the low copy repeats (LCRs) are depicted as gray boxes. The probes are depicted in colors corresponding to the FISH image. The 1.5-Mb region of chromosome 7q11.23 that is commonly deleted in WBS is indicated. Panel B shows a map of the region of exchange in block B leading to duplication of the common 1.5-Mb WBS deletion region. Polymorphic microsatellite markers and genes from the WBS region are shown on the top. The unique sequence is depicted by gray lines, and the repetitive blocks that constitute the LCRs are depicted as black arrows, indicating their orientation on the chromosome. A detailed schematic representation of the entire 143-kb length of block B is also shown. The dotted arrow on the right shows that the last 38 kb is absent in the centromeric B block (Bc). The genomic structure of genes within the region is also shown. The genes are those encoding general transcription factor 2 I (GTF2I), neutrophil cytosolic factor 1 (NCF1), and GTF2I repeat domain containing protein 2 (GTF2IRD2). Functional copies of these genes are present in block Bm, and corresponding pseudogenes (P1 or P2) are present in blocks Bc and Bt, except for the Bt copy of GTF2IRD2 that appears to be a functional gene as well. Exons are depicted as black boxes, and the numbers indicate the first and last exons within the block. Numerical identification, location, and sequence difference of each site-specific nucleotide (SSN) that was used in this study are indicated and have been described previously.4 All dosage-analysis calculations were performed with regard to Patient 1, who carried one normal chromosome 7, and his father, who carried two normal chromosomes 7. The inferred recombinant block B in the duplicated chromosome is shown at the bottom. The predicted genotypes at each position are depicted as circles: white circles denote Bc-type, black circles Bm-type; and black-and-white circles either Bc-type or Bm-type. The region of exchange occurred somewhere between SSN 4 and SSN 6 within the NCF1 gene and one of its pseudogenes.
Analysis of single-copy microsatellite markers from within the WBS region revealed that Patient 1 carried three distinct alleles at loci D7S2476, D7S3194, and D7S1870 (Table 1 of the Supplementary Appendix). In each case, only one of the alleles was present in the father, indicating that Patient 1 had inherited two different copies of the WBS region from his mother. Analysis of the maternal grandparents revealed that the duplicated chromosome in the proband contained segments of chromosome 7 that were inherited independently from each maternal grandparent (Table 1 and Figure 1 of the Supplementary Appendix). These analyses indicate that the meiotic interchromosomal recombination that led to the duplication took place in the mother's germ cells, making it a new rearrangement.
Reciprocal of the WBS Deletion
Further narrowing of the duplication breakpoints could not be accomplished with FISH or microsatellite analysis, owing to the high sequence identity of the LCRs. We used site-specific nucleotide (SSN) dosage analysis to define the relative number of B block–type copies (centromeric, medial, or telomeric) at each position analyzed, an approach that enabled us to infer the position where the recombination had occurred.4 Analysis of seven SSNs showed that in Patient 1 the transition between blocks Bm and Bc occurred between SSN 4 and SSN 6, within the NCF1 gene (Figure 2B). The majority of WBS deletion breakpoints (>90 percent) also occur within the B block,4 indicating that the duplication is the exact reciprocal of the common WBS deletion.
Altered Expression of Genes within the Duplication
Gene-expression analysis in lymphoblastoid cell lines by real-time PCR demonstrated that five of six genes that were examined within the duplicated region (GTF2I, LIMK1, WBSCR1, RFC2, and BAZ1B) showed increased expression in Patient 1 and reduced expression in persons with WBS (Table 2 of the Supplementary Appendix). The exception was WBSCR5, which showed reduced expression in persons with WBS but levels in Patient 1 that were consistent with levels in controls. WBSCR16, located just outside the telomeric deletion or duplication, did not show altered expression in either Patient 1 or in persons with WBS, indicating that the rearrangement breakpoints do not affect its transcription. The nearest gene outside the centromeric breakpoint, the gene encoding calneuron 1, was not expressed in lymphoblasts, but it is at least 300 kb from the proximal B block4 and is separated from the breakpoint by the complex and actively transcribed LCRs; hence, its expression unlikely to be affected.
Discussion
We describe a syndrome that is associated with a reciprocal duplication of the WBS microdeletion region. Patient 1 has intellectual strengths and weaknesses that are in direct contrast to those of children with WBS. Expressive language, especially syntax and phonology, is the area of greatest weakness for Patient 1, whereas for children with WBS, expressive language is a relative strength.2 When Patient 1 had difficulty making himself understood, he often successfully resorted to drawing what he was trying to express. Visuospatial construction (including drawing) is the area of greatest weakness for children with WBS, and most children with WBS who are Patient 1's age are able to draw only a few recognizable objects.15
Previous descriptions of persons with larger duplications of the region (supernumerary ring chromosome 7) have noted delay in expressive language or impairment accompanied by articulation problems, but none of these reports contain standardized assessment results or comparative data on expressive and receptive language.16 Karyotypes were derived from G-banding, not from molecular analysis, so the extent of each duplication remains unknown. Our findings specify the expressive-language phenotype associated with dup7q11.23 and define the precise region of chromosome 7 contributing to it as the 1.5-Mb interval commonly deleted in WBS.
There appears to be a subtle but recognizable facial phenotype that is shared by both Patient 1 and previously described persons with supernumerary ring chromosome 7, one that consists of a high and broad nose, posteriorly rotated ears, a high-arched palate, and a short philtrum.16,17,18 When accompanied by delay in expressive language, this gestalt warrants testing for duplication of the WBS critical region.
Although there is a strong genetic component to language impairment, so far only the transcription factor FOXP2 has been implicated as a cause of the problem, and only in a few cases.19 Disruption of FOXP2 results in reduced functional dosage and leads to deficits in both expressive and receptive language, in addition to orofacial dyspraxia that impairs the coordination of complex fine-motor movements of the lower face.19,20 The remaining cases of language impairment were predicted to involve complex causes.1 The identification of a second locus associated with language impairment is therefore unexpected and opens up the possibility of linking the expressive-language phenotype to a specific gene or genes from within the WBS region.
The contrast between phenotypes associated with deletion and those associated with duplication of the WBS region, as well as the accompanying changes in gene expression, suggests that genes within this region are dosage sensitive. These observations also suggests that perturbation can have a dramatic, negative effect on both language development and visuospatial construction ability.
The duplication or deletion encompasses 26 to 27 genes, many of which can be ruled out as important contributors to the WBS phenotype on the basis of the correlation of genotype with phenotype in persons with atypical deletions.21,22,23,24 The minimal critical interval that must be deleted in classic WBS spans the region between the gene encoding elastin and the common distal breakpoint and encompasses just nine genes. Included within the WBS minimal critical interval are three general transcription factor 2 I (GTF2I) genes, encoding a family of proteins that are predicted to possess some functional redundancy.25 In a simple or more complex model, alteration of expression of a single gene — or in the case of the GTF2I family, possibly a combination of genes — might lead to a distinctive language-impairment phenotype that overlaps with that observed in Patient 1. Our discovery of the WBS duplication provides both the long-sought-after reference case for ascertainment of other patients and an entry point toward the discovery of new genes required for the normal acquisition and expression of language. The association between expressive-language delay and duplication of the WBS region also has implications for the molecular diagnosis of language delay.
Supported by grants from the Canadian Institutes of Health Research and the Sick Kids Foundation (both to Dr. Osborne), the Spanish Ministries of Health and Science and Technology (to Dr. Pérez-Jurado), Genome Canada/Ontario Genomics Institute and Genome Spain (to Drs. Pérez-Jurado, Scherer, and Osborne), the National Institute of Neurological Disorders and Stroke (NS35102, to Drs. Mervis and Morris), and the National Institute of Child Health and Human Development (HD29957, to Dr. Mervis). Dr. Scherer is a Canadian Institutes of Health Research investigator and an international scholar of the Howard Hughes Medical Institute, and Dr. Osborne is a Canadian Institutes of Health Research scholar.
We are indebted to the patient and his family for their participation in our study and to the Centre for Applied Genomics at the Hospital for Sick Children, Toronto, for technical assistance.
Source Information
From the Department of Medical Genetics, University of Alberta, Edmonton, Alta., Canada (M.J.S., S.B., M.L.); the Department of Psychological and Brain Sciences, University of Louisville, Louisville, Ky. (C.B.M., E.P.); the Institute of Medical Science (E.J.Y., L.R.O.) and the Departments of Molecular and Medical Genetics (W.L., S.W.S., L.R.O.) and Medicine (L.R.O.), University of Toronto, and the Program in Genetics and Genomic Biology, Hospital for Sick Children (E.-J.S., S.W.S.) — both in Toronto; the Unitat de Genètica, Departament Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona (M.C., L.A.P.-J.); and the Department of Pediatrics, University of Nevada School of Medicine, Las Vegas (C.A.M.).
Drs. Somerville and Mervis contributed equally to this article.
Address reprint requests to Dr. Osborne at the Department of Medicine, University of Toronto, 7238 Medical Sciences Bldg., 1 King's College Circle, Toronto, ON M5S 1A8, Canada, or at lucy.osborne@utoronto.ca.
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