Supravalvar aortic stenosis (SVAS) is a characteristic feature of Williams-Beuren syndrome (WBS). SVAS is present in 67% of those with WBS, but severity varies; 21% have clinically significant SVAS requiring surgical intervention while 33% have no appreciable aortic disease. Little is known about genetic modifiers outside the 7q11.23 region that might contribute to SVAS severity. To investigate, we collaboratively phenotyped 473 individuals with WBS and performed the largest whole-genome- sequencing study to date. We developed a set of strategies for modifier discovery including extreme phenotyping (surgical SVAS vs. no SVAS) and prioritization of non-synonymous variants with increased predicted functional impact along with an allele frequency difference between the extreme phenotype groups. We identified pathways enriched in common or less frequent variants, followed by association testing of SVAS severity with the enriched pathways. The common variant analysis identified pathways including the extracellular matrix and the innate immune system, while pathways encompassing adaptive immunity, ciliary function, lipid metabolism and PI3KAKT were captured by both the common and less frequent variant analyses. Cell cycle and estrogen responsive pathways were among those identified through the less frequent variant analysis. Among the 69 genes reported in other large genome wide association studies assessing aortic traits, 11 genes, including PCSK9 and ILR6, were found in our study, suggesting overlapping disease mechanisms. In summary, this study presents novel strategies for identification of disease modifiers in rare conditions like WBS. ![Figure][1] ### Competing Interest Statement All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: BPR, CAM, and CBM have received funding from the WSA in the past. As a parent advocacy group, the WSA does have an interest in the submitted work but does not stand to financially profit from the findings. Additional government support noted above. No financial relationships with any other organizations that might have an interest in the submitted work occurred in the previous three years; no other relationships or activities that could appear to have influenced the submitted work are noted. ### Funding Statement The NIH effort was supported by the NHLBI Division of Intramural Research (BAK). BPR and CAM were supported by grants from the Williams Syndrome Association (WSA) and LRO received funding from the Canadian Institutes for Health Research (MOP77720. CBM received support from the National Institute of Neurological Disorders and Stroke (R01 NS35102) and the WSA (WSA 0104 and WSA 0111). CAM and LRO were also partially supported by subcontracts from the grant of R01 NS35102. The Genomic Disorder Biobank of the Telethon Network of Genetic Biobanks was supported by Telethon Italy grant GTB12001G, GM) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Review Board of the National Institutes of Health, the University of Nevada School of Medicine Internal Review Board, the University of Toronto Health Sciences Research Ethics Board, the Boston Children Hospital Internal Review Board, and Fondazione IRCCS Casa Sollievo della Sofferenza Ethics Board gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The summary statistics of the non-synonymous variants in no SVAS and surgical SVAS groups will be available upon reasonable request to the corresponding author, Dr. Beth Kozel via beth.kozel@nih.gov. [1]: pending:yes

Williams syndrome (WS), caused by microdeletion of some 21 genes on chromosome 7q11.23, is characterized by dysmorphic features, mental retardation or learning difficulties, elastin arteriopathy, and striking neurocognitive and social-behavioral abnormalities. Recent studies of murine knockouts of key genes in the microdeleted region, LIM kinase 1 (LIMK1) and cytoplasmatic linker protein 2 (CYLN2), demonstrated significant functional and metabolic abnormalities, but grossly normal structure, in the hippocampal formation (HF). Furthermore, deficits in spatial navigation and long-term memory, major cognitive domains dependent on hippocampal function, have been described in WS. We used multimodal neuroimaging to characterize hippocampal structure, function, and metabolic integrity in 12 participants with WS and 12 age-, sex-, and IQ-matched healthy controls. PET and functional MRI studies showed profound reduction in resting blood flow and absent differential response to visual stimuli in the anterior HF in WS. Spectroscopic measures of N-acetyl aspartate, considered a marker of synaptic activity, were reduced. Hippocampal size was preserved, but subtle alterations in shape were present. These data demonstrate abnormalities in HF in WS in agreement with murine models, implicate LIMK1 and CYLN2 in human hippocampal function, and suggest that hippocampal dysfunction may contribute to neurocognitive abnormalities in WS.

Knowledge of the environmental and genetic etiologies of complex cognitive disorders can guide strategies for diagnosis, prevention, and therapy, but disentangling the various causes can be very challenging. Two basic approaches can be used in identifying genetic factors. a top-down approach. in which phenotypic descriptions are used to discover genes that influence those phenotypes. and a bottom-up approach, comparing the phenotypic effects of genes that are known to cause syndromes that include cognitive disabilities. Thorough characterization of phenotypes throughout development is critical to both of these methods. These strategies have been applied to the characterization of genetic etiologies for reading disability, language disorders. attention-deficit hyperactivity disorder., and autism.

Molecular genetics advances have accelerated the discovery of the genetic etiology of many syndromes and diseases, and progress has been made in sorting out the causes of more complex traits such as diabetes and mental illness. Genotype-phenotype correlation study is a powerful method used to link the DNA code (genotype) with observable, measurable characteristics (the phenotype). The traditional method of gene discovery has proceeded from the phenotype to the genotype, but new techniques of gene manipulation have allowed experiments in the opposite direction-from genotype to phenotype. This reverse genetics includes knocking out a candidate gene in an animal and measuring the effects on the organism and its development. In experimental animals, the gene can be knocked out in different strains, which also allows study of gene expression clues regarding modifying genes. In humans, genotype to phenotype study is possible when a rare chromosome anomaly such as a translocation or inversion disrupts a single gene, providing a candidate gene for the phenotype. Unlike animal studies, however, humans with particular genetic mutations or deletions do not have the same genetic background or environment, potentially clouding the interpretation of the phenotype. Many of these chromosomal syndromes include common complex conditions such as mental retardation as part of the phenotype. Discovery of a gene accounting or predisposing for mental retardation in a particular syndrome likely identifies one of the quantitative trait loci in the general population. Further, such an advance may lead to the discovery of a family of genes or a biologic pathway that contributes to normal neurodevelopment. However, the individual gene would probably account for only a small percentage of the variability of the trait in the general population with nonsyndromic mental retardation.

Most individuals with Williams syndrome (WS) have a 1.6 Mb deletion in chromosome 7q11.23 that encompasses the elastin ( ELN ) gene, while most families with autosomal dominant supravalvar aortic stenosis (SVAS) have point mutations in ELN . The overlap of the clinical phenotypes of the two conditions (cardiovascular disease and connective tissue abnormalities such as hernias) is due to the effect of haploinsufficiency of ELN . SVAS families often have affected individuals with some WS facial features, most commonly in infancy, suggesting that ELN plays a role in WS facial gestalt as well. To find other genes contributing to the WS phenotype, we studied five families with SVAS who have small deletions in the WS region. None of the families had mental retardation, but affected family members had the Williams Syndrome Cognitive Profile (WSCP). All families shared a deletion of LIMK1 , which encodes a protein strongly expressed in the brain, supporting the hypothesis that LIMK1 hemizygosity contributes to impairment in visuospatial constructive cognition. While the deletions from the families nearly spanned the WS region, none had a deletion of FKBP6 or GTF2I , suggesting that the mental retardation seen in WS is associated with deletion of either the centromeric and/or telomeric portions of the region. Comparison of these five families with reports of other individuals with partial deletions of the WS region most strongly implicates GTF2I in the mental retardation of WS. © 2003 Wiley‐Liss, Inc.

Heat shock protein 27 (HSP27) is one of a number of actin‐binding proteins that regulate actin polymerization. Three related HSP27 sequences had previously been mapped to chromosomes 3, 9, and X. We have used fluorescent in‐situ hybridization (FISH) to correct and refine the map position of the transcribed HSP27 gene (locus HSPB1) to chromosome 7q11.23. This band also contains the site of the deletion associated with Williams syndrome (WS). To define the relationship between HSP27 and the WS deletion, we used two‐color FISH on previously G‐banded and photographed metaphase chromosomes from WS cell‐lines and peripheral blood. Six WS patients with longer deletions that extend telomeric to the classical WS deletion region were analyzed for deletion length using HSP27, cosmids generated from P193O22 (cos11) and B350L10 (cos64 and 82), B350L10, B161A02, and B363M4. The BAC 363M4 was selected from the Washington University database and contains HSP27. Our results indicated that HSP27 was deleted in three patients and that HSP27 is telomeric to cos11, cos64, cos82, and B350L10. B363M4 was demonstrated to overlap the telomeric end of B161A02 and HSP27 may be contained partially within the telomeric end of B161A02. The possible role of HSP27 in the cognitive features of WS is discussed. © 2003 Wiley‐Liss, Inc.

OBJECTIVESThe purpose was to demonstrate that an objective, multivariate case definition of fetal alcohol syndrome (FAS) and partial fetal alcohol syndrome (PFAS) can be derived by means of craniofacial anthropometry.STUDY DESIGNCraniofacial measurements (n = 21) were taken of 100 individuals who had been exposed to alcohol before birth (41 FAS, 59 PFAS) and 31 members of a control group. Multivariate discriminant analysis was used to identify craniofacial measurements that best differentiated the 3 groups (FAS, PFAS, and control).RESULTSBoth the FAS and PFAS groups had a unique craniofacial phenotype that could be accurately discriminated from that of the control group. Stepwise discriminant analysis identified 6 craniofacial measurements that could differentiate individuals with and without prenatal alcohol exposure with 96% accuracy, 98% sensitivity, and 90% specificity.CONCLUSIONSIndividuals with FAS and PFAS have a distinctive facial phenotype that can be characterized anthropometrically. The phenotypic case definition could be used as a screening tool to identify individuals prenatally exposed to alcohol who do not exhibit a "classic" FAS phenotype but exhibit a more subtle craniofacial dysmorphia.

The 27-kDa human heat shock protein (hsp27) is expressed in a wide variety of tissues in the absence of stress and is thought to regulate actin filament dynamics. Three related human hsp27 sequences had been previously mapped to chromosomes 3, 9, and X (McGuire et al., 1989). We have used fluorescent in situ hybridization (FISH) to correct and refine the map position of the transcribed hsp27 gene to chromosome 7, band 7q11.23 and its two pseudogenes to chromosome bands 9q21 and Xp11.2. Band 7q11.23 is the site of a deletion associated with Williams syndrome (WS), a congenital developmental disorder involving the vascular, connective tissue, and central nervous systems. We performed FISH mapping of hsp27 DNA to the chromosomes of four WS patients and found that in the majority of cells (77%), a bright signal was found on only one chromosome 7 at band 7q11.23. These results may indicate that the WS deletion includes the gene for the 27-kDa heat shock protein. However, in a small proportion of cells, a second, less intense signal was found on the other chromosome 7. We have interpreted these second weak signals as cross-hybridization due to the repeated DNA sequences (Alu sequences) known to reside within the introns and the flanking regions of this gene. Southern hybridization analysis is currently being performed to substantiate these findings in WS patient DNAs.REFERENCE: McGuire SE, Fuqua SA, Naylor SL, Helin-Davis DA, McGuire WL (1989): Chromosomal assignments of human 27-kDa heat shock protein gene family Somat Cell Mol Genet 15:167-71.

Williams syndrome (WS) is a developmental disorder caused by deletion of multiple genes at chromosome 7q11.23. Here, we report the identification and characterization of a novel gene,WSTF,that maps to the common WS deletion region.WSTFencodes a novel protein of 1425 amino acids with unknown function. It contains one PHD-type zinc finger motif followed by a bromodomain. Both motifs are found in many transcription regulators, suggesting thatWSTFmay function as a transcription factor.WSTFis ubiquitously expressed in both adult and fetal tissues. TheWSTFgene consists of 20 exons spanning about 80 kb. Fluorescencein situhybridization analysis shows thatWSTFis deleted in 50/50 WS individuals. Hemizygous deletion ofWSTFmay contribute to WS.

Supravalvular aortic stenosis (SVAS) is an inherited vascular disease that can cause heart failure and death. SVAS can be inherited as an autosomal dominant trait or as part of a developmental disorder, Williams syndrome (WS). In recent studies we presented evidence suggesting that a translocation disrupting the elastin gene caused SVAS in one family while deletions involving the entire elastin locus caused WS. In this study, pulsed-field, PCR, and Southern analyses showed that a 100-kb deletion of the 3' end of the elastin gene cosegregated with the disease in another SVAS family. DNA sequence analysis localized the breakpoint between elastin exons 27 and 28, the same region disrupted by the SVAS-associated translocation. These data indicate that mutations in the elastin gene cause SVAS and suggest that elastin exons 28-36 may encode critical domains for vascular development.

Crouzon craniofacial dysostosis (CFD) is an autosomal dominant craniofacial disorder characterized by premature craniosynostosis, shallow orbits and hypoplastic maxilla. To map the gene responsible, we have used a mapping strategy of testing for linkage to known developmental genes. Analysis of a large kindred established linkage between CFD and three loci (D10S190, D10S209 and D10S216) that span a 13 cM region on chromosome 10q. A maximum pairwise lod score of 4.42 (theta = 0) at D10S190 was obtained and the addition of a second kindred produced a combined pairwise lod score of 5.32 (theta = 0) at the same locus. The developmental gene, PAX2, located within this region, is an attractive candidate gene.