We describe 19 unrelated individuals with submicroscopic deletions involving 10p15.3 characterized by chromosomal microarray (CMA). Interestingly, to our knowledge, only two individuals with isolated, submicroscopic 10p15.3 deletion have been reported to date; however, only limited clinical information is available for these probands and the deleted region has not been molecularly mapped. Comprehensive clinical history was obtained for 12 of the 19 individuals described in this study. Common features among these 12 individuals include: cognitive/behavioral/developmental differences (11/11), speech delay/language disorder (10/10), motor delay (10/10), craniofacial dysmorphism (9/12), hypotonia (7/11,), brain anomalies (4/6) and seizures (3/7). Parental studies were performed for nine of the 19 individuals; the 10p15.3 deletion was de novo in seven of the probands, not maternally inherited in one proband and inherited from an apparently affected mother in one proband. Molecular mapping of the 19 individuals reported in this study has identified two genes, ZMYND11 (OMIM# 608668) and DIP2C (OMIM# 611380) (UCSC Genome Browser), mapping within 10p15.3 which are most commonly deleted. Although no single gene has been identified which is deleted in all 19 individuals studied, the deleted region in all but one individual includes ZMYND11 and the deleted region in all but one other individual includes DIP2C. There is not a clearly identifiable phenotypic difference between these two individuals and the size of the deleted region does not generally predict clinical features. Little is currently known about these genes complicating a direct genotype/phenotype correlation at this time. These data however, suggest that ZMYND11 and/or DIP2C haploinsufficiency contributes to the clinical features associated with 10p15 deletions in probands described in this study.
chromosomal microarray (CMA); 10p15.3; deletion; ZMYND11; DIP2C
Cornelia de Lange Syndrome (CdLS) is a multisystem developmental disorder characterized by growth retardation, cognitive impairment, external and internal structural malformations, and characteristic facial features. Currently, there are no definitive prenatal screening measures that lead to the diagnosis of CdLS. In this study, documented prenatal findings in CdLS syndrome were analyzed towards the development of a prenatal profile predictive of CdLS. We reviewed 53 cases of CdLS (29 previously reported and 24 unreported) in which prenatal observations/findings were available. The review of these cases revealed a pattern of sonographic findings, including obvious associated structural defects, growth restriction, as well as a more subtle, but strikingly characteristic, facial profile, suggestive of a recognizable prenatal ultrasonographic profile for CdLS. In addition the maternal serum marker, PAPP-A, may be reduced and fetal nuchal translucency may be increased in some pregnancies when measured at an appropriate gestational age. In conclusion, CdLS can be prenatally diagnosed or readily ruled out in a family with a known mutation in a CdLS gene. The characteristic ultrasonographic profile may allow for prenatal diagnosis of CdLS in 1) subsequent pregnancies to a couple with a prior child with CdLS in whom a mutation has not been identified or 2) when there are unexplained pregnancy signs of fetal abnormality such as oligo- or polyhydramnios, a low maternal serum PAPP-A level and/or increased nuchal translucency, fetal growth retardation, or structural anomalies consistent with CdLS.
PAPP-A; IUGR; Cornelia de Lange Syndrome; CdLS; Prenatal screening
Cornelia de Lange syndrome (CdLS) is a genetic disorder associated with delayed growth, intellectual disability, limb reduction defects and characteristic facial features. Germline mosaicism has been a described mechanism for CdLS when there are several affected offspring of apparently unaffected parents. Presently, the recurrence risk for CdLS has been estimated to be as high as 1.5%; however, this figure may be an underrepresentation. We report on the molecularly defined germline mosaicism cases from a large CdLS database, representing the first large case series on germline mosaicism in CdLS. Of the 12 families, eight have been previously described; however, four have not. No one specific gene mutation, either in the NIPBL or the SMC1A gene, was associated with an increased risk for germline mosaicism. Suspected or confirmed cases of germline mosaicism in our database range from a conservative 3.4% up to 5.4% of our total cohort. In conclusion, the potential reproductive recurrence risk due to germline mosiacism should be addressed in prenatal counseling for all families who have had a previously affected pregnancy or child with CdLS.
Cornelia de Lange syndrome; Germline Mosaicism; Germ Cell; Mosaicism; Genetic Counseling
Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder caused by mutations in the cohesin-loading protein NIPBL1,2 for nearly 60% of individuals with classical CdLS3-5 and in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands6,7. In humans, the multi-subunit complex cohesin is comprised of SMC1, SMC3, RAD21 and a STAG protein to form a ring structure proposed to encircle sister chromatids to mediate sister chromatid cohesion (SCC)8 as well as play key roles in gene regulation9. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin10-13 and in yeast, HOS1, a class I histone deacetylase, deacetylates SMC3 during anaphase14-16. Here we report the identification of HDAC8 as the vertebrate SMC3 deacetylase as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation (SMC3-ac) and inefficient dissolution of the “used” cohesin complex released from chromatin in both prophase and anaphase. While SMC3 with retained acetylation is loaded onto chromatin, ChIP-Seq analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.
Cornelia de Lange syndrome (CdLS; or Brachmann-de Lange syndrome) is a dominantly inherited congenital malformation disorder with features that include characteristic facies, cognitive delays, growth retardation and limb anomalies. Mutations in nearly 60% of CdLS patients have been identified in NIPBL, which encodes a regulator of the sister chromatid cohesion complex. NIPBL, also known as delangin, is a homolog of yeast and amphibian Scc2 and C. elegans PQN-85. Although the exact mechanism of NIPBL function in sister chromatid cohesion is unclear, in vivo yeast and C. elegans experiments and in vitro vertebrate cell experiments have demonstrated that NIPBL/Scc2 functionally interacts with the MAU2/Scc4 protein to initiate loading of cohesin onto chromatin. To test the significance of this model in the clinical setting of CdLS, we fine-mapped the NIBPL–MAU2 interaction domain and tested the functional significance of missense mutations and variants in NIPBL and MAU2 identified in these minimal domains in a cohort of patients with CdLS. We demonstrate that specific novel mutations at the N-terminus of the MAU2-interacting domain of NIBPL result in markedly reduced MAU2 binding, although we appreciate no consistent clinical difference in the small group of patients with these mutations. These data suggest that factors in addition to MAU2 are essential in determining the clinical features and severity of CdLS.
Cornelia de Lange syndrome; cohesin; NIPBL; MAU2; SCC4; sister chromatid cohesion
Hearing loss is the most prevalent sensory perception deficit in humans, affecting 1/500 newborns, can be syndromic or nonsyndromic and is genetically heterogeneous. Nearly 80% of inherited nonsyndromic bilateral sensorineural hearing loss (NBSNHI) is autosomal recessive. Although many causal genes have been identified, most are minor contributors, except for GJB2, which accounts for nearly 50% of all recessive cases of severe to profound congenital NBSNHI in some populations. More than 60% of children with a NBSNHI do not have an identifiable genetic cause. To identify genetic contributors, we genotyped 659 GJB2 mutation negative pediatric probands with NBSNHI and assayed for copy number variants (CNVs). After identifying 8 mild-moderate NBSNHI probands with a Chr15q15.3 deletion encompassing the Stereocilin (STRC) gene amongst this cohort, sequencing of STRC was undertaken in these probands as well as 50 probands and 14 siblings with mild-moderate NBSNHI and 40 probands with moderately severe-profound NBSNHI who were GJB2 mutation negative. The existence of a STRC pseudogene that is 99.6% homologous to the STRC coding region has made the sequencing interpretation complicated. We identified 7/50 probands in the mild-moderate cohort to have biallelic alterations in STRC, not including the 8 previously identified deletions. We also identified 2/40 probands to have biallelic alterations in the moderately severe-profound NBSNHI cohort, notably no large deletions in combination with another variant were found in this cohort. The data suggest that STRC may be a common contributor to NBSNHI among GJB2 mutation negative probands, especially in those with mild to moderate hearing impairment.
Bilateral sensorineural hearing loss; SNHI; Chr15q15.3; Stereocilin; STRC; DFNB16; SNP genotyping array; copy number variation; CNV
Cornelia de Lange syndrome (CdLS) is a congenital disorder characterized by distinctive facial features, growth retardation, limb abnormalities, intellectual disability, and behavioral problems. Autism has been reported to occur frequently in CdLS, but the frequency of autism in individuals with the milder CdLS phenotype is not well studied. We investigated autistic features by using a screening tool and a diagnostic interview in 49 individuals with the mild to moderate phenotype from a CdLS research database at the Children’s Hospital of Philadelphia. The Social Communication Questionnaire (SCQ), a screening instrument for autistic disorder, was completed for all individuals. For individuals who screened positive and a subset of those that screened negative, the Autism Diagnostic Interview-Revised (ADI-R) was administered. Autistic symptom severity was not significantly different by gender, age groups, and genotypes. There was a significant correlation between higher levels of adaptive functioning and lower scores of autistic symptoms. The estimated prevalence of significant autistic features by ADI-R criteria was 43% in our cohort of individuals with the mild to moderate CdLS phenotype, which suggests that prevalence of autistic disorder may be higher than previously described among individuals with mild to moderate phenotype of CdLS. Clinicians who take care of individuals with CdLS should have a high index of suspicion for autistic features, and refer for further evaluation when these features are present in order to expedite appropriate intervention.
Cornelia de Lange syndrome (CdLS); autism; behavioral phenotype; NIPBL; SMC1A; SMC3
Congenital heart disease (CHD) has been reported to occur in 14–70% of individuals with Cornelia de Lange syndrome (CdLS, OMIM 122470) and accounts for significant morbidity and mortality when present. Charts from a cohort of 479 patients with CdLS were reviewed for cardiac evaluations, gene testing and information to determine phenotypic severity. Two hundred fifty-nine individuals had either documented structural defects or minor cardiac findings. The presence of CHD was then quantified as a function of mutation status and severity of CdLS: mild, moderate, or severe. Different types of CHD were also evaluated by mutation status to assess for any genotype –phenotype correlation. NIPBL, SMC1A, and SMC3 mutation-positive patients were equally likely to have CHD, although the number of SMC1A and SMC3 mutation-positive patients were small in comparison. Structural CHDs were more likely to be present in individuals with moderate and severe CdLS than in the mild phenotype. This study evaluates the trends of CHD seen in the CdLS population and correlates these findings with genotype.
Cornelia de Lange syndrome (CdLS); congenital heart disease (CHD); mutation; phenotype; cohesin; NIPBL; SMC1A; SMC3
To identify causes of death (COD) in propositi with Cornelia de Lange syndrome (CdLS) at various ages, and to develop guidelines to improve management and avoid morbidity and mortality, we retrospectively reviewed a total of 426 propositi with confirmed clinical diagnoses of CdLS in our database who died in a 41-year period between 1966–2007. Of these, 295 had an identifiable COD reported to us. Clinical, laboratory and complete autopsy data were completed on 41, of which 38 were obtainable, an additional 19 had autopsies that only documented the COD, and 45 propositi had surgical, imaging, or terminal event clinical documentation of their COD. Proband ages ranged from fetuses (21 to 40 weeks gestation) to 61 years. A literature review was undertaken to identify all reported causes of death in CdLS individuals.
In our cohort of 295 propositi with a known COD, respiratory causes including aspiration/reflux and pneumonias were the most common primary causes (31%), followed by gastrointestinal disease, including obstruction/volvulus (19%). Congenital anomalies accounted for 15% of deaths and included congenital diaphragmatic hernia and congenital heart defects. Acquired cardiac disease accounted for 3% of deaths. Neurological causes and accidents each accounted for 8%, sepsis for 4%, cancer for 2%, renal disease for 1.7%, and other causes, 9% of deaths. We also present 21 representative clinical cases for illustration.
This comprehensive review has identified important etiologies contributing to the morbidity and mortality in this population that will provide for an improved understanding of clinical complications, and management for children and adults with CdLS.
Cornelia de Lange Syndrome; Brachmann de Lange Syndrome; causes of death; mortality; autopsy; CdLS
Thrombocytopenia was first reported in Cornelia de Lange Syndrome (CdLS) by Froster in 1993. Despite early reports, thrombocytopenia has been rarely reported in this disorder. We performed a retrospective analysis of a large cohort of patients with CdLS. We calculated prevalence of thrombocytopenia in 3 subsets of this cohort: the entire cohort (n=1740), a subset of subjects with substantial clinical records (n=695) and a subset of subjects with clinical information regarding platelet counts (n=85). This analysis revealed that 15 have had thrombocytopenia (18% of those with available blood counts); seven had Immune ThrombocytoPenia (ITP). The reported prevalence of pediatric ITP is between 5-13 per 100,000 persons. The prevalence of ITP in this cohort is between 7/1740 and 7/85, giving a relative risk of ITP of between 30 (CI 12 to 77) and 633 (CI 259-1549). Contrary to the reported cases in the literature, none of our patients have had progression of the thrombocytopenia nor have they developed other cytopenias. All 15 patients with thromobocytopenia had CdLS based on clinical criteria. Of the 10 patients tested for mutations in NIBPL, eight had mutations identified. These data support an increased incidence of thrombocytopenia and ITP in CdLS. Subsequently, patients are at risk for spontaneous hemorrhage, and likely increased risk secondary to the high frequency of self-injurious behavior. Although further studies are needed to better define the scope of the problem and to define the mechanisms of thrombocytopenia in CdLS, we would recommend screening for thrombocytopenia upon diagnosis and at five-year intervals thereafter.
cornelia de lange; thrombocytopenia; ITP; immune thrombocytopenia
Two brothers, with dissimilar clinical features, were each found to have different abnormalities of chromosome 20 by subtelomere fluorescence in situ hybridization (FISH). The proband had deletion of 20p subtelomere and duplication of 20q subtelomere, while his brother was found to have a duplication of 20p subtelomere and deletion of 20q subtelomere. Parental cytogenetic studies were initially thought to be normal, both by G-banding and by subtelomere FISH analysis. Since chromosome 20 is a metacentric chromosome and an inversion was suspected, we used anchored FISH to assist in identifying a possible inversion. This approach employed concomitant hybridization of a FISH probe to the short (p) arm of chromosome 20 with the 20q subtelomere probe. We identified a cytogenetically non-visible, mosaic pericentric inversion of one of the maternal chromosome 20 homologues, providing a mechanistic explanation for the chromosomal abnormalities present in these brothers. Array comparative genomic hybridization (CGH) with both a custom-made BAC and cosmid-based subtelomere specific array (TEL array) and a commercially-available SNP-based array confirmed and further characterized these rearrangements, identifying this as the largest pericentric inversion of chromosome 20 described to date. TEL array data indicate that the 20p breakpoint is defined by BAC RP11-978M13, ~900 kb from the pter; SNP array data reveal this breakpoint to occur within BAC RP11-978M13. The 20q breakpoint is defined by BAC RP11-93B14, ~1.7 Mb from the qter, by TEL array; SNP array data refine this breakpoint to within a gap between BACs on the TEL array (i.e. between RP11-93B14 and proximal BAC RP11-765G16).
FISH; subtelomere; array comparative genomic hybridization; array CGH; SNP; 20p; 20q; pericentric inversion; duplication; deletion
Cornelia de Lange Syndrome (CdLS) is a dominantly inherited heterogeneous genetic disorder with multi-system abnormalities. 60% of probands with CdLS have heterozygous mutations in the Nipped-B- like (NIPBL) gene, 5% have mutations in the SMC1A gene, and 1 proband was found to have a mutation in the SMC3 gene. Cohesin is a multi-subunit complex consisting of a SMC1A and SMC3 heterodimer and two non-SMC subunits. SMC1A is located on the human X chromosome and is reported to escape X inactivation. We show that 29 unrelated CdLS probands with 21 unique SMC1A mutations have been identified by our group and others including 7 males. All mutations identified to date are either missense or small deletions with all presumably preserving the protein open reading frame. Both wild type and mutant alleles are expressed. Females quantitatively express twice the amount of SMC1A mRNA as compared to males. The transcriptional profiling of 23 selected genes is different in SMC1A mutant probands, controls and NIPBL mutant probands. These results suggest that mechanistically SMC1A-related CdLS is not due to altered levels of the SMC1A transcript, but rather that the mutant proteins maintain a residual function in males and enact a dominant negative effect in females.
SMC1A; CdLS; X-linked; Expression
The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS.
The use of array technology to define chromosome deletions and duplications is bringing us closer to establishing a genotype/phenotype map of genomic copy number alterations. We studied 21 patients and 5 relatives with deletions of the short arm of chromosome 20 using the Illumina HumanHap550 SNP array to 1) more accurately determine the deletion sizes, 2) identify and compare breakpoints, 3) establish genotype/phenotype correlations and 4) investigate the use of the HumanHap550 platform for analysis of chromosome deletions. Deletions ranged from 95kb to 14.62Mb, and all of the breakpoints were unique. Eleven patients had deletions between 95kb and 4Mb and these individuals had normal development, with no anomalies outside of those associated with Alagille syndrome. The proximal and distal boundaries of these eleven deletions constitute a 5.4MB region, and we propose that haploinsufficiency for only 1 of the 12 genes in this region causes phenotypic abnormalities. This defines the JAG1 associated critical region, in which deletions do not confer findings other than those associated with Alagille syndrome. The other 10 patients had deletions between 3.28Mb and 14.62Mb, which extended outside the critical region, and notably, all of these patients, had developmental delay. This group had other findings such as autism, scoliosis and bifid uvula. We identified 47 additional polymorphic genome-wide copy number variants (>20 SNPs), with 0–5 variants called per patient. Deletions of the short arm of chromosome 20 are associated with relatively mild and limited clinical anomalies. The use of SNP arrays provides accurate high-resolution definition of genomic abnormalities.
SNP array analysis; 20p deletion; copy number variants; Alagille syndrome; haploinsufficiency; JAG1
Genome-wide studies using cells from patients with Cornelia de Lange Syndrome reveal a role for cohesin in regulating gene expression in human cells.
Cohesin regulates sister chromatid cohesion during the mitotic cell cycle with Nipped-B-Like (NIPBL) facilitating its loading and unloading. In addition to this canonical role, cohesin has also been demonstrated to play a critical role in regulation of gene expression in nondividing cells. Heterozygous mutations in the cohesin regulator NIPBL or cohesin structural components SMC1A and SMC3 result in the multisystem developmental disorder Cornelia de Lange Syndrome (CdLS). Genome-wide assessment of transcription in 16 mutant cell lines from severely affected CdLS probands has identified a unique profile of dysregulated gene expression that was validated in an additional 101 samples and correlates with phenotypic severity. This profile could serve as a diagnostic and classification tool. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.
Appropriate segregation of chromosomes to daughter cells depends upon proper cohesion of sister chromatids during mitosis. The multiprotein cohesin complex and its regulators are key factors in this process. Intriguingly, recent work has shown that the cohesin complex also has other cellular roles, including a role in regulating gene expression. Additionally, mutations in cohesin structural and regulatory components have been linked to human multisystem developmental disorders such as Cornelia de Lange Syndrome (CdLS), but the role cohesin is playing in the pathogenesis of this disorder is unknown. To define the role that cohesin plays in regulating gene expression in human cells, we analyzed gene expression and genome-wide cohesin binding patterns in cells from normal subjects and from CdLS probands with mutations in the cohesin regulator NIPBL or in the cohesin structural component SMC1A. We found a strikingly conserved pattern of gene dysregulation in these different cell lines that correlates with disease severity and a significant correlation between gene dysregulation and cohesin binding around misexpressed genes. The observed pattern of binding and misexpression is consistent with cohesin having a putative role as a boundary/insulator interacting protein or transcription factor, the activity of which is disrupted in CdLS probands.