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1.  Homozygosity for the V37I GJB2 Mutation in Fifteen Probands with Mild to Moderate Sensorineural Hearing Impairment: Further Confirmation of Pathogenicity and Haplotype Analysis in Asian Populations 
Hearing impairment affects 1 in 650 newborns, making it the most common congenital sensory impairment. Autosomal recessive nonsyndromic sensorineural hearing impairment (ARNSHI) comprises 80% of familial hearing impairment cases. Mutations in GJB2 account for a significant number of ARNSHI (and up to 50% of documented recessive (e.g. more than 1 affected sibling) hearing impairment in some populations). Mutations in the GJB2 gene are amongst the most common causes of hearing impairment in populations of various ethnic backgrounds. Two mutations of this gene, 35delG and 167delT, account for the majority of reported mutations in Caucasian populations, especially those of Mediterranean and Ashkenazi Jewish background. The 235delC mutation is most prevalent in East Asian populations. Some mutations are of less well characterized significance. The V37I missense mutation, common in Asian populations, was initially described as a polymorphism and later as a potentially pathogenic mutation. We report here on 15 unrelated individuals with ARNSHI and homozygosity for the V37I GJB2 missense mutation. Nine individuals are of Chinese ancestry, two are of unspecified Asian descent, one is of Japanese descent, one individual is of Vietnamese ancestry, one of Philippine background and one of Italian and Cuban/Caucasian background. Homozygosity for the V37I GJB2 mutation may be a more common pathogenic missense mutation in Asian populations, resulting in mild to moderate sensorineural hearing impairment. We report a presumed haplotype block specific to East Asian individuals with the V37I mutation encompassing the GJB2 gene that may account for the high prevalence in East Asian populations.
PMCID: PMC3745519  PMID: 23873582
GJB2; V37I; China; haplotype; hearing impairment
2.  PECONPI: A Novel Software for Uncovering Pathogenic Copy Number Variations in Non-Syndromic Sensorineural Hearing Loss and Other Genetically Heterogeneous Disorders 
This report describes an algorithm developed to predict the pathogenicity of copy number variants (CNVs) in large sample cohorts. CNVs (genomic deletions and duplications) are found in healthy individuals and in individuals with genetic diagnoses, and differentiation of these two classes of CNVs can be challenging and usually requires extensive manual curation. We have developed PECONPI, an algorithm to assess the pathogenicity of CNVs based on gene content and CNV frequency. This software was applied to a large cohort of patients with genetically heterogeneous non-syndromic hearing loss to score and rank each CNV based on its relative pathogenicity. Of 636 individuals tested, we identified the likely underlying etiology of the hearing loss in 14 (2%) of the patients (1 with a homozygous deletion, 7 with a deletion of a known hearing loss gene and a point mutation on the trans allele and 6 with a deletion larger than 1 Mb). We also identified two probands with smaller deletions encompassing genes that may be functionally related to their hearing loss. The ability of PECONPI to determine the pathogenicity of CNVs was tested on a second genetically heterogenous cohort with congenital heart defects (CHDs). It successfully identified a likely etiology in 6 of 355 individuals (2%). We believe this tool is useful for researchers with large genetically heterogeneous cohorts to help identify known pathogenic causes and novel disease genes.
PMCID: PMC3745548  PMID: 23897863
Copy number variation; genetic heterogeneity
3.  Prenatal Profile of Cornelia de Lange Syndrome (CdLS): A Review of 53 Pregnancies 
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.
PMCID: PMC3402646  PMID: 22740382
PAPP-A; IUGR; Cornelia de Lange Syndrome; CdLS; Prenatal screening
4.  Renal Anomalies in Alagille Syndrome: A Disease-Defining Feature 
Alagille syndrome (ALGS) is an autosomal dominant condition, primarily caused by mutations in JAGGED1. ALGS is defined by cholestatic liver disease, cardiac disease and involvement of the face, skeleton and eyes with variable expression of these features. Renal involvement has been reported though not formally described. The objective of this study was to systematically characterize the renal involvement in ALGS.
We performed a retrospective review of 466 JAGGED1 mutation-positive ALGS patients. Charts were reviewed for serum biochemistries, renal ultrasounds or other imaging, urinalysis and clinical reports from pediatric nephrologists. The clinical data were reviewed by two pediatric hepatologists and a pediatric nephrologist.
Of 466 charts reviewed we found 187 yielded evaluable renal information. Of these, 73/187 were shown to have renal involvement, representing 39% of the study cohort. Renal dysplasia was the most common anomaly seen. Genotype analysis of the JAGGED1 mutations in the patients with and without renal involvement did not reveal an association with mutation type.
From the study we concluded that renal involvement has a prevalence of 39% in ALGS in our evaluable patients. Renal dysplasia is the most common renal anomaly. This finding correlates with the known role of the Notch pathway in glomerular development. Since renal disease of the type seen in ALGS can impair growth and impact liver transplantation, there is a clear need for a prospective study of renal involvement in ALGS and the development of guidelines for evaluation and management. These data also suggest that renal involvement be considered the sixth defining criterion for ALGS.
PMCID: PMC4511708  PMID: 22105858
liver disease; renal disease; Alagille syndrome kidney
5.  Utility and limitations of exome sequencing as a genetic diagnostic tool for conditions associated with pediatric sudden cardiac arrest/sudden cardiac death 
Human Genomics  2015;9(1):15.
Conditions associated with sudden cardiac arrest/death (SCA/D) in youth often have a genetic etiology. While SCA/D is uncommon, a pro-active family screening approach may identify these inherited structural and electrical abnormalities prior to symptomatic events and allow appropriate surveillance and treatment. This study investigated the diagnostic utility of exome sequencing (ES) by evaluating the capture and coverage of genes related to SCA/D.
Samples from 102 individuals (13 with known molecular etiologies for SCA/D, 30 individuals without known molecular etiologies for SCA/D and 59 with other conditions) were analyzed following exome capture and sequencing at an average read depth of 100X. Reads were mapped to human genome GRCh37 using Novoalign, and post-processing and analysis was done using Picard and GATK. A total of 103 genes (2,190 exons) related to SCA/D were used as a primary filter. An additional 100 random variants within the targeted genes associated with SCA/D were also selected and evaluated for depth of sequencing and coverage. Although the primary objective was to evaluate the adequacy of depth of sequencing and coverage of targeted SCA/D genes and not for primary diagnosis, all patients who had SCA/D (known or unknown molecular etiologies) were evaluated with the project’s variant analysis pipeline to determine if the molecular etiologies could be successfully identified.
The majority of exons (97.6 %) were captured and fully covered on average at minimum of 20x sequencing depth. The proportion of unique genomic positions reported within poorly covered exons remained small (4 %). Exonic regions with less coverage reflect the need to enrich these areas to improve coverage. Despite limitations in coverage, we identified 100 % of cases with a prior known molecular etiology for SCA/D, and analysis of an additional 30 individuals with SCA/D but no known molecular etiology revealed a diagnostic answer in 5/30 (17 %). We also demonstrated 95 % of 100 randomly selected reported variants within our targeted genes would have been picked up on ES based on our coverage analysis.
ES is a helpful clinical diagnostic tool for SCA/D given its potential to successfully identify a molecular diagnosis, but clinicians should be aware of limitations of available platforms from technical and diagnostic perspectives.
Electronic supplementary material
The online version of this article (doi:10.1186/s40246-015-0038-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4506570  PMID: 26187847
6.  Genome-wide SNP Genotyping Identifies the Stereocilin (STRC) Gene as a Major Contributor to Pediatric Bilateral Sensorineural Hearing Impairment 
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.
PMCID: PMC3264741  PMID: 22147502
Bilateral sensorineural hearing loss; SNHI; Chr15q15.3; Stereocilin; STRC; DFNB16; SNP genotyping array; copy number variation; CNV
7.  Congenital Heart Disease in Cornelia de Lange Syndrome: Phenotype and Genotype Analysis 
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.
PMCID: PMC3551981  PMID: 22965847
Cornelia de Lange syndrome (CdLS); congenital heart disease (CHD); mutation; phenotype; cohesin; NIPBL; SMC1A; SMC3
8.  Causes of Death and Autopsy Findings in a Large Study Cohort of Individuals with Cornelia de Lange Syndrome and Review of the Literature 
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.
PMCID: PMC3222915  PMID: 22069164
Cornelia de Lange Syndrome; Brachmann de Lange Syndrome; causes of death; mortality; autopsy; CdLS
9.  Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance 
Kaiser, Frank J. | Ansari, Morad | Braunholz, Diana | Concepción Gil-Rodríguez, María | Decroos, Christophe | Wilde, Jonathan J. | Fincher, Christopher T. | Kaur, Maninder | Bando, Masashige | Amor, David J. | Atwal, Paldeep S. | Bahlo, Melanie | Bowman, Christine M. | Bradley, Jacquelyn J. | Brunner, Han G. | Clark, Dinah | Del Campo, Miguel | Di Donato, Nataliya | Diakumis, Peter | Dubbs, Holly | Dyment, David A. | Eckhold, Juliane | Ernst, Sarah | Ferreira, Jose C. | Francey, Lauren J. | Gehlken, Ulrike | Guillén-Navarro, Encarna | Gyftodimou, Yolanda | Hall, Bryan D. | Hennekam, Raoul | Hudgins, Louanne | Hullings, Melanie | Hunter, Jennifer M. | Yntema, Helger | Innes, A. Micheil | Kline, Antonie D. | Krumina, Zita | Lee, Hane | Leppig, Kathleen | Lynch, Sally Ann | Mallozzi, Mark B. | Mannini, Linda | Mckee, Shane | Mehta, Sarju G. | Micule, Ieva | Mohammed, Shehla | Moran, Ellen | Mortier, Geert R. | Moser, Joe-Ann S. | Noon, Sarah E. | Nozaki, Naohito | Nunes, Luis | Pappas, John G. | Penney, Lynette S. | Pérez-Aytés, Antonio | Petersen, Michael B. | Puisac, Beatriz | Revencu, Nicole | Roeder, Elizabeth | Saitta, Sulagna | Scheuerle, Angela E. | Schindeler, Karen L. | Siu, Victoria M. | Stark, Zornitza | Strom, Samuel P. | Thiese, Heidi | Vater, Inga | Willems, Patrick | Williamson, Kathleen | Wilson, Louise C. | Hakonarson, Hakon | Quintero-Rivera, Fabiola | Wierzba, Jolanta | Musio, Antonio | Gillessen-Kaesbach, Gabriele | Ramos, Feliciano J. | Jackson, Laird G. | Shirahige, Katsuhiko | Pié, Juan | Christianson, David W. | Krantz, Ian D. | Fitzpatrick, David R. | Deardorff, Matthew A.
Human Molecular Genetics  2014;23(11):2888-2900.
Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.
PMCID: PMC4014191  PMID: 24403048
10.  Two Siblings with Alternate Unbalanced Recombinants Derived from a Large Cryptic Maternal Pericentric Inversion of Chromosome 20 
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).
PMCID: PMC2840621  PMID: 20101690
FISH; subtelomere; array comparative genomic hybridization; array CGH; SNP; 20p; 20q; pericentric inversion; duplication; deletion
11.  SMC1A Expression and Mechanism of Pathogenicity in Probands with X-linked Cornelia de Lange Syndrome 
Human mutation  2009;30(11):1535-1542.
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.
PMCID: PMC2783874  PMID: 19701948
SMC1A; CdLS; X-linked; Expression
12.  Cohesin embraces new phenotypes 
Nature genetics  2014;46(11):1157-1158.
A new study identifies homozygous missense mutations in SGOL1, which encodes a component of the cohesin complex, in a newly described disorder termed Chronic Atrial and Intestinal Dysrhythmia (CAID) syndrome. These findings implicate cohesin in the regulation of intrinsic cardiac and intestinal rhythm and further expand the growing group of disorders termed the cohesinopathies.
PMCID: PMC4268132  PMID: 25352100
13.  Precocious Sister Chromatid Separation (PSCS) in Cornelia de Lange Syndrome 
The Cornelia de Lange syndrome (CdLS) (OMIM# 122470) is a dominantly inherited multisystem developmental disorder. The phenotype consists of characteristic facial features, hirsutism, abnormalities of the upper extremities ranging from subtle changes in the phalanges and metacarpal bones to oligodactyly and phocomelia, gastroesophageal dysfunction, growth retardation, and neurodevelopmental delay. Prevalence is estimated to be as high as 1 in 10,000. Recently, mutations in NIPBL were identified in sporadic and familial CdLS cases. To date, mutations in this gene have been identified in over 45% of individuals with CdLS. NIPBL is the human homolog of the Drosophila Nipped-B gene. Although its function in mammalian systems has not yet been elucidated, sequence homologs of Nipped-B in yeast (Scc2 and Mis4) are required for sister chromatid cohesion during mitosis, and a similar role was recently demonstrated for Nipped-B in Drosophila. In order to evaluate NIPBL role in sister chromatid cohesion in humans, metaphase spreads on 90 probands (40 NIPBL mutation positive and 50 NIPBL mutation negative) with CdLS were evaluated for evidence of precocious sister chromatid separation (PSCS). We screened 50 metaphases from each proband and found evidence of PSCS in 41% (compared to 9% in control samples). These studies indicate that NIPBL may play a role in sister chromatid cohesion in humans as has been reported for its homologs in Drosophila and yeast.
PMCID: PMC2766539  PMID: 16100726
cornelia de Lange syndrome; CdLS; NIPBL; Nipped-B; precocious sister chromatid separation; PSCS
14.  Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome 
PLoS ONE  2014;9(10):e108853.
Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.
PMCID: PMC4199614  PMID: 25329894
15.  Facial Diagnosis of Mild and Variant CdLS: Insights From a Dysmorphologist Survey 
Cornelia de Lange syndrome (CdLS) is a dominant disorder with classic severe forms and milder atypical variants. Central to making the diagnosis is identification of diagnostic facial features. With the recognition that patients with SMC1A and SMC3 mutations have milder, atypical features, we surveyed 65 dysmorphologists using facial photographs from 32 CdLS patients with the goals of (1) Illustrating examples of milder patients with SMC1A mutations and (2) Obtaining objective data to determine which facial features were useful and misleading in making a diagnosis of CdLS. Clinicians were surveyed whether the patient had CdLS or another diagnosis, the certainty of response and the clinical features used to support each response. Using only facial photographs, an average of 24 cases (75%) were accurately diagnosed per clinician. Correct diagnoses were made in 90% of classic CdLS and 87% of non-CdLS cases, however, only 54% of mild or variant CdLS were correctly diagnosed by respondents. We confirmed that CdLS is most accurately diagnosed in childhood and the diagnosis becomes increasingly difficult with age. This survey demonstrated that emphasis is placed on the eyebrows, nasal features, prominent upper lip and micrognathia. In addition, the presence of fuller, atypical eyebrows, a prominent nasal bridge and significant prognathism with age dissuaded survey takers from arriving at a diagnosis of CdLS in individuals with mild NIPBL and SMC1A mutations. This work underscores the difficulty in diagnosing patients with mild and variant CdLS and serves to objectively classify both useful and misleading features in the diagnosis of CdLS.
PMCID: PMC4133091  PMID: 20583156
Cornelia de Lange syndrome; Brachmann–de Lange syndrome; facial features; facies; NIPBL; SMC1; SMC1A; SMC1L1; SMC3; mild; severe; survey; dysmorphology
16.  Clinical phenotype-based gene prioritization: an initial study using semantic similarity and the human phenotype ontology 
BMC Bioinformatics  2014;15(1):248.
Exome sequencing is a promising method for diagnosing patients with a complex phenotype. However, variant interpretation relative to patient phenotype can be challenging in some scenarios, particularly clinical assessment of rare complex phenotypes. Each patient’s sequence reveals many possibly damaging variants that must be individually assessed to establish clear association with patient phenotype. To assist interpretation, we implemented an algorithm that ranks a given set of genes relative to patient phenotype. The algorithm orders genes by the semantic similarity computed between phenotypic descriptors associated with each gene and those describing the patient. Phenotypic descriptor terms are taken from the Human Phenotype Ontology (HPO) and semantic similarity is derived from each term’s information content.
Model validation was performed via simulation and with clinical data. We simulated 33 Mendelian diseases with 100 patients per disease. We modeled clinical conditions by adding noise and imprecision, i.e. phenotypic terms unrelated to the disease and terms less specific than the actual disease terms. We ranked the causative gene against all 2488 HPO annotated genes. The median causative gene rank was 1 for the optimal and noise cases, 12 for the imprecision case, and 60 for the imprecision with noise case. Additionally, we examined a clinical cohort of subjects with hearing impairment. The disease gene median rank was 22. However, when also considering the patient’s exome data and filtering non-exomic and common variants, the median rank improved to 3.
Semantic similarity can rank a causative gene highly within a gene list relative to patient phenotype characteristics, provided that imprecision is mitigated. The clinical case results suggest that phenotype rank combined with variant analysis provides significant improvement over the individual approaches. We expect that this combined prioritization approach may increase accuracy and decrease effort for clinical genetic diagnosis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-248) contains supplementary material, which is available to authorized users.
PMCID: PMC4117966  PMID: 25047600
Clinical; Phenotype; Exome; Genome; Informatics
17.  Semaphorin 3d signaling defects are associated with anomalous pulmonary venous connections 
Nature medicine  2013;19(6):760-765.
Total anomalous pulmonary venous connection (TAPVC) is a potentially lethal congenital disorder that occurs when the pulmonary veins do not connect normally to the left atrium, allowing mixing of pulmonary and systemic blood1. In contrast to the extensive knowledge of arterial vascular patterning, little is known about the patterning of veins. Here we show that the secreted guidance molecule semaphorin 3d (Sema3d) is crucial for the normal patterning of pulmonary veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand (MES), the precursor of the common pulmonary vein, does not form at the proper location on the dorsal surface of the embryonic common atrium2,3. However, we found that TAPVC occurs in Sema3d mutant mice despite normal formation of the MES. In these embryos, the maturing pulmonary venous plexus does not anastomose uniquely with the properly formed MES. In the absence of Sema3d, endothelial tubes form in a region that is normally avascular, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary. Sequencing of SEMA3D in individuals with anomalous pulmonary veins identified a phenylalanine-to-leucine substitution that adversely affects SEMA3D function. These results identify Sema3d as a crucial pulmonary venous patterning cue and provide experimental evidence for an alternate developmental model to explain abnormal pulmonary venous connections.
PMCID: PMC3746328  PMID: 23685842
18.  A Cohesin-Independent Role for NIPBL at Promoters Provides Insights in CdLS 
PLoS Genetics  2014;10(2):e1004153.
The cohesin complex is crucial for chromosome segregation during mitosis and has recently also been implicated in transcriptional regulation and chromatin architecture. The NIPBL protein is required for the loading of cohesin onto chromatin, but how and where cohesin is loaded in vertebrate cells is unclear. Heterozygous mutations of NIPBL were found in 50% of the cases of Cornelia de Lange Syndrome (CdLS), a human developmental syndrome with a complex phenotype. However, no defects in the mitotic function of cohesin have been observed so far and the links between NIPBL mutations and the observed developmental defects are unclear. We show that NIPBL binds to chromatin in somatic cells with a different timing than cohesin. Further, we observe that high-affinity NIPBL binding sites localize to different regions than cohesin and almost exclusively to the promoters of active genes. NIPBL or cohesin knockdown reduce transcription of these genes differently, suggesting a cohesin-independent role of NIPBL for transcription. Motif analysis and comparison to published data show that NIPBL co-localizes with a specific set of other transcription factors. In cells derived from CdLS patients NIPBL binding levels are reduced and several of the NIPBL-bound genes have previously been observed to be mis-expressed in CdLS. In summary, our observations indicate that NIPBL mutations might cause developmental defects in different ways. First, defects of NIPBL might lead to cohesin-loading defects and thereby alter gene expression and second, NIPBL deficiency might affect genes directly via its role at the respective promoters.
Author Summary
The cohesin complex is crucial for chromosome segregation during cell divisions but was recently also implicated in transcriptional regulation and chromatin architecture. Cohesin's binding to chromatin depends on NIPBL, a factor that was found to be mutated in 50% of the cases of the human developmental disorder Cornelia de Lange Syndrome (CdLS). To understand the role of NIPBL for cohesin, we need to know when and where the cohesin is loaded onto DNA. Our experiments have identified high-affinity NIPBL binding sites in different cells lines which do not overlap with cohesin-binding, but colocalize with specific transcription factors at active promoters. The activity of the respective genes depends on NIPBL but not cohesin. This is in contrast with other published data showing colocalization of NIPBL and cohesin, and we reveal the existence of different types of NIPBL binding sites that are detected differently by the antibodies used in the different studies. Our observations reveal a dual role for NIPBL in cohesin loading and as potential transcription co-factor, which yields novel insights into how NIPBL defects could cause Cornelia de Lange Syndrome since NIPBL mutations might directly influence developmentally important genes.
PMCID: PMC3923681  PMID: 24550742
19.  Mutation Spectrum and Genotype–Phenotype Correlation in Cornelia de Lange Syndrome 
Human mutation  2013;34(12):10.1002/humu.22430.
Cornelia de Lange syndrome (CdLS) is a clinically and genetically heterogeneous developmental disorder. Clinical features include growth retardation, intellectual disability, limb defects, typical facial dysmorphism, and other systemic involvement. The increased understanding of the genetic basis of CdLS has led to diagnostic improvement and expansion of the phenotype. Mutations in five genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8), all regulators or structural components of cohesin, have been identified. Approximately 60% of CdLS cases are due to NIPBL mutations, 5% caused by mutations in SMC1A, RAD21, and HDAC8 and one proband was found to carry a mutation in SMC3. To date, 311 CdLS-causing mutations are known including missense, nonsense, small deletions and insertions, splice site mutations, and genomic rearrangements. Phenotypic variability is seen both intra- and intergenically. This article reviews the spectrum of CdLS mutations with a particular emphasis on their correlation to the clinical phenotype.
PMCID: PMC3880228  PMID: 24038889
Cornelia de Lange syndrome; NIPBL; SMC1A; SMC3; RAD21; HDAC8
20.  Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes 
Journal of proteome research  2012;11(12):6111-6123.
Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS.
PMCID: PMC3519430  PMID: 23106691
Cohesin; Cornelia de Lange syndrome; SMC1A; SMC3; 2D-DIGE; proteomic profile; dysregulated protein expression; c-Myc
21.  Subtelomeric Deletion of Chromosome 10p15.3: Clinical Findings and Molecular Cytogenetic Characterization 
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.
PMCID: PMC3429713  PMID: 22847950
chromosomal microarray (CMA); 10p15.3; deletion; ZMYND11; DIP2C
22.  NOTCH2 mutations in Alagille syndrome 
Journal of medical genetics  2011;49(2):138-144.
Alagille syndrome (ALGS) is a dominant, multisystem disorder caused by mutations in the Jagged1 (JAG1) ligand in 94% of patients, and in the NOTCH2 receptor in <1%. There are only two NOTCH2 families reported to date. This study hypothesised that additional NOTCH2 mutations would be present in patients with clinical features of ALGS without a JAG1 mutation.
The study screened a cohort of JAG1-negative individuals with clinical features suggestive or diagnostic of ALGS for NOTCH2 mutations.
Eight individuals with novel NOTCH2 mutations (six missense, one splicing, and one non-sense mutation) were identified. Three of these patients met classic criteria for ALGS and five patients only had a subset of features. The mutations were distributed across the extracellular (N=5) and intracellular domains (N=3) of the protein. Functional analysis of four missense, one nonsense, and one splicing mutation demonstrated decreased Notch signalling of these proteins. Subjects with NOTCH2 mutations demonstrated highly variable expressivity of the affected systems, as with JAG1 individuals. Liver involvement was universal in NOTCH2 probands and they had a similar prevalence of ophthalmologic and renal anomalies to JAG1 patients. There was a trend towards less cardiac involvement in the NOTCH2 group (60% vs 100% in JAG1). NOTCH2 (+) probands exhibited a significantly decreased penetrance of vertebral abnormalities (10%) and facial features (20%) when compared to the JAG1 (+) cohort.
This work confirms the importance of NOTCH2 as a second disease gene in ALGS and expands the repertoire of the NOTCH2 related disease phenotype.
PMCID: PMC3682659  PMID: 22209762
23.  Clinical Report: Germline Mosaicism in Cornelia de Lange Syndrome 
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.
PMCID: PMC3356507  PMID: 22581668
Cornelia de Lange syndrome; Germline Mosaicism; Germ Cell; Mosaicism; Genetic Counseling
24.  HDAC8 mutations in Cornelia de Lange Syndrome affect the cohesin acetylation cycle 
Nature  2012;489(7415):313-317.
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.
PMCID: PMC3443318  PMID: 22885700

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