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1.  Development and clinical application of an integrative genomic approach to personalized cancer therapy 
Genome Medicine  2016;8:62.
Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics.
We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data.
We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases.
These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-016-0313-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4888213  PMID: 27245685
Cancer; Genomics; Personalized medicine; Clinical application
2.  A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects 
PLoS Genetics  2016;12(4):e1005848.
Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.
Author Summary
Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities in the central nervous system (CNS). Patients affected with gLEs have brain white matter defects that can be seen on MRI and exhibit variable neurologic phenotypes including motor impairment, hypotonia, pyramidal dysfunction, dystonia and/or dyskinesias, ataxia, seizures, cortical blindness, optic atrophy, and impaired cognitive development. The exact etiology of half of gLEs is unknown. We studied three unrelated families affected with an undiagnosed gLE and discovered a homozygous germline mutation c.2536T>G in VPS11, a gene involved in membrane trafficking and fusion of lysosomes and endosomes, as a novel cause of a new gLE syndrome. The mutation in VPS11 results in protein instability and impaired protein complex assembly. In addition, we show that VPS11 is required for proper autophagic activities in human cells. Importantly, we characterized a zebrafish line carrying a vps11 mutation and confirmed its essential role in brain white matter development and neuron survival.
PMCID: PMC4847778  PMID: 27120463
3.  Evaluation of the Affymetrix CytoScan® Dx Assay for Developmental Delay 
The goal of molecular cytogenetic testing for children presenting with developmental delay is to identify or exclude genetic abnormalities that are associated with cognitive, behavioral, and/or motor symptoms. Until 2010, chromosome analysis was the standard first-line genetic screening test for evaluation of patients with developmental delay when a specific syndrome was not suspected. In 2010, The American College of Medical Genetics and several other groups recommended chromosomal microarray (CMA) as the first-line test in children with developmental delays, multiple congenital anomalies, and/or autism. This test is able to detect regions of genomic imbalances at a much finer resolution than G-banded karyotyping. Until recently, no CMA testing had been approved by the United States Food and Drug Administration (FDA). This review will focus on the use of the Affymetrix CytoScan® Dx Assay, the first CMA to receive FDA approval for the genetic evaluation of individuals with developmental delay.
PMCID: PMC4415685  PMID: 25350348
Affymetrix CytoScan® Dx Assay; global developmental delay; mental retardation; chromosomal microarray; genetic testing
4.  Myeloid dysregulation in a human induced pluripotent stem cell model of PTPN11-associated juvenile myelomonocytic leukemia 
Cell reports  2015;13(3):504-515.
Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germ-line PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and up-regulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were up-regulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations but not those without PTPN11 defects. Reducing miR-223’s function in NS/JMML hiPSCs normalized myelogenesis. Micro-RNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover micro-RNA dysregulation, establishing a genotype-phenotype association for JMML and providing novel therapeutic targets.
Graphical abstract
PMCID: PMC4618050  PMID: 26456833
5.  A PTPN11 allele encoding a catalytically impaired SHP2 protein in a patient with a Noonan syndrome phenotype 
The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser) and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy’s phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity.
PMCID: PMC4134745  PMID: 24891296
double mutation; LEOPARD; Noonan; PTPN11
6.  ClinLabGeneticist: a tool for clinical management of genetic variants from whole exome sequencing in clinical genetic laboratories 
Genome Medicine  2015;7(1):77.
Routine clinical application of whole exome sequencing remains challenging due to difficulties in variant interpretation, large dataset management, and workflow integration. We describe a tool named ClinLabGeneticist to implement a workflow in clinical laboratories for management of variant assessment in genetic testing and disease diagnosis. We established an extensive variant annotation data source for the identification of pathogenic variants. A dashboard was deployed to aid a multi-step, hierarchical review process leading to final clinical decisions on genetic variant assessment. In addition, a central database was built to archive all of the genetic testing data, notes, and comments throughout the review process, variant validation data by Sanger sequencing as well as the final clinical reports for future reference. The entire workflow including data entry, distribution of work assignments, variant evaluation and review, selection of variants for validation, report generation, and communications between various personnel is integrated into a single data management platform. Three case studies are presented to illustrate the utility of ClinLabGeneticist. ClinLabGeneticist is freely available to academia at
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0207-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4558641  PMID: 26338694
7.  46,XY disorder of sex development and developmental delay associated with a novel 9q33.3 microdeletion encompassing NR5A1 
European journal of medical genetics  2013;56(11):10.1016/j.ejmg.2013.09.006.
Steroidogenic factor 1 (SF1) is a nuclear receptor encoded by the NR5A1 gene. SF1 affects both sexual and adrenal development through the regulation of target gene expression. Genotypic male and female SF1 knockout mice have adrenal and gonadal agenesis with persistent Müllerian structures and early lethality. There have been several reports of NR5A1 mutations in individuals with 46,XY complete gonadal dysgenesis (CGD) or other disorders of sex development (DSD) with or without an adrenal phenotype. To date microdeletions involving NR5A1 have been reported in only two patients with DSDs. We report a novel microdeletion encompassing NR5A1 in a patient with 46,XY DSD and developmental delay. The phenotypically female patient initially presented with mild developmental delay and dysmorphisms. Chromosome analysis revealed a 46,XY karyotype. A 1.54 Mb microdeletion of chromosome 9q33.3 including NR5A1 was detected by array CGH and confirmed by FISH. Normal maternal FISH results indicated that this was most likely a de novo event. Since most NR5A1 mutations have been ascertained through gonadal or adrenal abnormalities, the additional findings of developmental delay and minor facial dysmorphisms are possibly related to haploinsufficiency of other genes within the 1.54 Mb deleted region. This report further confirms the role of NR5A1 deletions in 46,XY DSD and reinforces the utility of aCGH in the work up of DSDs of unclear etiology.
PMCID: PMC3880784  PMID: 24056159
NR5A1; SF1; 46,XY disorder of sex development; 46,XY DSD; 9q33 microdeletion
8.  The Impact of CNVs on Outcomes for Infants with Single Ventricle Heart Defects 
Human genomes harbor copy number variants (CNVs), regions of DNA gains or losses. While pathogenic CNVs are associated with congenital heart disease (CHD), their impact on clinical outcomes is unknown. This study sought to determine whether pathogenic CNVs among infants with single ventricle (SV) physiology were associated with inferior neurocognitive and somatic growth outcomes.
Methods and Results
Genomic DNAs from 223 subjects of two National Heart, Lung, and Blood Institute-sponsored randomized clinical trials with infants with SV CHD and 270 controls from The Cancer Genome Atlas project were analyzed for rare CNVs >300 kb using array comparative genomic hybridization. Neurocognitive and growth outcomes at 14 months from the CHD trials were compared among subjects with and without pathogenic CNVs. Putatively pathogenic CNVs, comprising 25 duplications and 6 deletions, had a prevalence of 13.9%, significantly greater than the 4.4% rate of such CNVs among controls. CNVs associated with genomic disorders were found in 13 cases but no control. Several CNVs likely to be causative of SV CHD were observed, including aberrations altering the dosage of GATA4, MYH11, and GJA5. Subjects with pathogenic CNVs had worse linear growth, and those with CNVs associated with known genomic disorders had the poorest neurocognitive and growth outcomes. A minority of children with pathogenic CNVs were noted to be dysmorphic on clinical genetics examination.
Pathogenic CNVs appear to contribute to the etiology of SV forms of CHD in at least 10% of cases, are clinically subtle but adversely affect outcomes in children harboring them.
PMCID: PMC3987966  PMID: 24021551
copy number variant; congenital cardiac defect; outcome; hypoplastic left heart syndrome
9.  Multi-ethnic Cytochrome-P450 Copy Number Profiling: Novel Pharmacogenetic Alleles and Mechanism of Copy Number Variation Formation 
The pharmacogenomics journal  2012;13(6):10.1038/tpj.2012.48.
To determine the role of CYP450 copy number variation (CNV) beyond CYP2D6, 11 CYP450 genes were interrogated by MLPA and qPCR in 542 African-American, Asian, Caucasian, Hispanic, and Ashkenazi Jewish individuals. The CYP2A6, CYP2B6 and CYP2E1 combined deletion/duplication allele frequencies ranged from 2% to 10% in these populations. High-resolution microarray-based comparative genomic hybridization (aCGH) localized CYP2A6, CYP2B6 and CYP2E1 breakpoints to directly-oriented low-copy repeats. Sequencing localized the CYP2B6 breakpoint to a 529 bp intron 4 region with high homology to CYP2B7P1, resulting in the CYP2B6*29 partial deletion allele and the reciprocal, and novel, CYP2B6/2B7P1 duplicated fusion allele (CYP2B6*30). Together, these data identified novel CYP450 CNV alleles (CYP2B6*30 and CYP2E1*1Cx2) and indicate that common CYP450 CNV formation is likely mediated by non-allelic homologous recombination resulting in both full gene and gene-fusion copy number imbalances. Detection of these CNVs should be considered when interrogating these genes for pharmacogenetic drug selection and dosing.
PMCID: PMC3580117  PMID: 23164804
CYP2A6; CYP2B6; CYP2E1; copy number variation; pharmacogenetics; pharmacogenomics
10.  Analytical validation of whole exome and whole genome sequencing for clinical applications 
BMC Medical Genomics  2014;7:20.
Whole exome and genome sequencing (WES/WGS) is now routinely offered as a clinical test by a growing number of laboratories. As part of the test design process each laboratory must determine the performance characteristics of the platform, test and informatics pipeline. This report documents one such characterization of WES/WGS.
Whole exome and whole genome sequencing was performed on multiple technical replicates of five reference samples using the Illumina HiSeq 2000/2500. The sequencing data was processed with a GATK-based genome analysis pipeline to evaluate: intra-run, inter-run, inter-mode, inter-machine and inter-library consistency, concordance with orthogonal technologies (microarray, Sanger) and sensitivity and accuracy relative to known variant sets.
Concordance to high-density microarrays consistently exceeds 97% (and typically exceeds 99%) and concordance between sequencing replicates also exceeds 97%, with no observable differences between different flow cells, runs, machines or modes. Sensitivity relative to high-density microarray variants exceeds 95%. In a detailed study of a 129 kb region, sensitivity was lower with some validated single-base insertions and deletions “not called”. Different variants are "not called" in each replicate: of all variants identified in WES data from the NA12878 reference sample 74% of indels and 89% of SNVs were called in all seven replicates, in NA12878 WGS 52% of indels and 88% of SNVs were called in all six replicates. Key sources of non-uniformity are variance in depth of coverage, artifactual variants resulting from repetitive regions and larger structural variants.
PMCID: PMC4022392  PMID: 24758382
11.  Phelan-McDermid syndrome: a review of the literature and practice parameters for medical assessment and monitoring 
Autism spectrum disorder (ASD) and intellectual disability (ID) can be caused by mutations in a large number of genes. One example is SHANK3 on the terminal end of chromosome 22q. Loss of one functional copy of SHANK3 results in 22q13 deletion syndrome or Phelan-McDermid syndrome (PMS) and causes a monogenic form of ASD and/or ID with a frequency of 0.5% to 2% of cases. SHANK3 is the critical gene in this syndrome, and its loss results in disruption of synaptic function. With chromosomal microarray analyses now a standard of care in the assessment of ASD and developmental delay, and with the emergence of whole exome and whole genome sequencing in this context, identification of PMS in routine clinical settings will increase significantly. However, PMS remains a rare disorder, and the majority of physicians have never seen a case. While there is agreement about core deficits of PMS, there have been no established parameters to guide evaluation and medical monitoring of the syndrome. Evaluations must include a thorough history and physical and dysmorphology examination. Neurological deficits, including the presence of seizures and structural brain abnormalities should be assessed as well as motor deficits. Endocrine, renal, cardiac, and gastrointestinal problems all require assessment and monitoring in addition to the risk of recurring infections, dental and vision problems, and lymphedema. Finally, all patients should have cognitive, behavioral, and ASD evaluations. The objective of this paper is to address this gap in the literature and establish recommendations to assess the medical, genetic, and neurological features of PMS.
PMCID: PMC4362650  PMID: 25784960
Phelan-McDermid syndrome; 22q13 deletion syndrome; SHANK3; Autism; Autism spectrum disorder; Neurodevelopmental disorders; Practice parameters
12.  Localization of BRCA1 protein in breast cancer tissue and cell lines with mutations 
The breast and ovarian cancer susceptibility gene (BRCA1) encodes a tumor suppressor. The BRCA1 protein is found primarily in cell nuclei and plays an important role in the DNA damage response and transcriptional regulation. Deficiencies in DNA repair capabilities have been associated with higher histopathological grade and worse prognosis in breast cancer.
In order to investigate the subcellular distribution of BRCA1 in tumor tissue we randomly selected 22 breast carcinomas and tested BRCA1 protein localization in frozen and contiguous formalin-fixed, paraffin embedded (FFPE) tissue, using pressure cooker antigen-retrieval and the MS110 antibody staining. To assess the impact of BRCA1 germline mutations on protein localization, we retrospectively tested 16 of the tumor specimens to determine whether they contained the common Ashkenazi Jewish founder mutations in BRCA1 (185delAG, 5382insC), and BRCA2 (6174delT). We also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC).
In FFPE tissue, with MS110 antibody staining, we frequently found reduced BRCA1 nuclear staining in breast tumor tissue compared to normal tissue, and less BRCA1 staining with higher histological grade in the tumors. However, in the frozen sections, BRCA1 antibody staining showed punctate, intra-nuclear granules in varying numbers of tumor, lactating, and normal cells. Two mutation carriers were identified and were confirmed by gene sequencing. We have also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC) and found altered sub-nuclear and nucleolar localization patterns consistent with a functional impact of the mutation on protein localization.
The data presented here support a role for BRCA1 in the pathogenesis of sporadic and inherited breast cancers. The use of well-characterized reagents may lead to further insights into the function of BRCA1 and possibly the further development of targeted therapeutics.
PMCID: PMC3720266  PMID: 23855721
Breast cancer; BRCA1 mutations; Frozen section immunohistology; Nucleolar localization
13.  Copy number variation and warfarin dosing: evaluation of CYP2C9, VKORC1, CYP4F2, GGCX and CALU 
Pharmacogenomics  2011;13(3):297-307.
To determine if copy number variants contribute to warfarin dose requirements, we investigated CYP2C9, VKORC1, CYP4F2, GGCX and CALU for deletions and duplications in a multiethnic patient population treated with therapeutic doses of warfarin.
Patients & methods
DNA samples from 178 patients were subjected to copy number analyses by multiplex ligation-dependent probe amplification or quantitative PCR assays. Additionally, the CYP2C9 exon 8 insertion/deletion polymorphism (rs71668942) was examined among the patient cohort and 1750 additional multiethnic healthy individuals.
All patients carried two copies of CYP2C9 by multiplex ligation-dependent probe amplification and no exon 8 deletion carriers were detected. Similarly, quantitative PCR assays for VKORC1, CYP4F2, GGCX and CALU identified two copies in all populations.
These data indicate that copy number variants in the principal genes involved in warfarin dose variability (CYP2C9, VKORC1), including genes with lesser effect (CYP4F2, GGCX), and those that may be more relevant among certain racial groups (CALU), are rare in multiethnic populations, including African–Americans.
PMCID: PMC3292047  PMID: 22188360
CALU; CNV; copy number variation; CYP2C9; CYP4F2; GGCX; pharmacogenetics; VKORC1; warfarin
14.  Experience with Carrier Screening and Prenatal Diagnosis for Sixteen Ashkenazi Jewish Genetic Diseases 
Human mutation  2010;31(11):1240-1250.
The success of prenatal carrier screening as a disease prevention strategy in the Ashkenazi Jewish (AJ) population has driven the expansion of screening panels as disease-causing founder mutations have been identified. However, the carrier frequencies of many of these mutations have not been reported in large AJ cohorts. We determined the carrier frequencies of over 100 mutations for 16 recessive disorders in the New York metropolitan area AJ population. Among the 100% AJ-descended individuals, screening for 16 disorders resulted in ~1 in 3.3 being a carrier for one disease and ~1 in 24 for two diseases. The carrier frequencies ranged from 0.066 (1 in 15.2; Gaucher disease) to 0.006 (1 in 168; nemaline myopathy), which averaged ~15% higher than those for all screenees. Importantly, over 95% of screenees chose to be screened for all possible AJ diseases, including disorders with lower carrier frequencies and/or detectability. Carrier screening also identified rare individuals homozygous for disease-causing mutations who had previously unrecognized clinical manifestations. Additionally, prenatal testing results and experience for all 16 disorders (n = 574) are reported. Together, these data indicate the general acceptance, carrier frequencies, and prenatal testing results for an expanded panel of 16 diseases in the AJ population.
PMCID: PMC2970726  PMID: 20672374
Ashkenazi Jewish; carrier screening; carrier frequency; residual risk; prenatal diagnosis
15.  Large inverted repeats within Xp11.2 are present at the breakpoints of isodicentric X chromosomes in Turner syndrome 
Human Molecular Genetics  2010;19(17):3383-3393.
Turner syndrome (TS) results from whole or partial monosomy X and is mediated by haploinsufficiency of genes that normally escape X-inactivation. Although a 45,X karyotype is observed in half of all TS cases, the most frequent variant TS karyotype includes the isodicentric X chromosome alone [46,X,idic(X)(p11)] or as a mosaic [46,X,idic(X)(p11)/45,X]. Given the mechanism of idic(X)(p11) rearrangement is poorly understood and breakpoint sequence information is unknown, this study sought to investigate the molecular mechanism of idic(X)(p11) formation by determining their precise breakpoint intervals. Karyotype analysis and fluorescence in situ hybridization mapping of eight idic(X)(p11) cell lines and three unbalanced Xp11.2 translocation lines identified the majority of breakpoints within a 5 Mb region, from ∼53 to 58 Mb, in Xp11.1–p11.22, clustering into four regions. To further refine the breakpoints, a high-resolution oligonucleotide microarray (average of ∼350 bp) was designed and array-based comparative genomic hybridization (aCGH) was performed on all 11 idic(X)(p11) and Xp11.2 translocation lines. aCGH analyses identified all breakpoint regions, including an idic(X)(p11) line with two potential breakpoints, one breakpoint shared between two idic(X)(p11) lines and two Xp translocations that shared breakpoints with idic(X)(p11) lines. Four of the breakpoint regions included large inverted repeats composed of repetitive gene clusters and segmental duplications, which corresponded to regions of copy-number variation. These data indicate that the rearrangement sites on Xp11.2 that lead to isodicentric chromosome formation and translocations are probably not random and suggest that the complex repetitive architecture of this region predisposes it to rearrangements, some of which are recurrent.
PMCID: PMC2916707  PMID: 20570968
17.  Type 1 Gaucher Disease: Significant disease manifestations in “asymptomatic” homozygotes identified by prenatal carrier screening 
Archives of internal medicine  2010;170(16):1463-1469.
Type 1 Gaucher Disease (GD), an autosomal recessive lysosomal storage disease, is most prevalent in the Ashkenazi Jewish (AJ) population. Experts have suggested that up to two-thirds of AJ homozygotes for the common mutation (N370S) are asymptomatic throughout life and never come to medical attention. However, there are no systematic studies of N370S homozygotes to support this presumption.
Prenatal carrier screening of 8069 AJ adults for six common GD mutations was performed. GD manifestations in 37 previously unrecognized homozygotes were assessed by clinical, laboratory and imaging studies.
Among the 8069 AJ screenees, 524 GD carriers (1:15.4) and nine previously unrecognized GD homozygotes (1:897) were identified, consistent with that expected (1:949, p=1.0). Six of these homozygotes, and 31 AJ GD homozygotes identified by other prenatal carrier screening programs in the New York metropolitan area were evaluated (aged 17-40 years). Of these, 84% were N370S homozygotes, others being heteroallelic for N370S and V394L, L444P or R496H. Notably, 65% reported no GD medical complaints. However, 49% had anemia and/or thrombocytopenia. Among the 29 who had imaging studies, 97% had mild to moderate splenomegaly and 55% had hepatomegaly; skeletal imaging revealed marrow infiltration (100%), Erlenmeyer flask deformities (43%), lucencies (22%) and bone infarcts (14%). DEXA studies of 25 homozygotes found 60% osteopenic or osteoporotic.
Contrary to previous discussions, almost all asymptomatic GD homozygotes serendipitously diagnosed by prenatal carrier screening had disease manifestations and should be followed for disease progression and institution of appropriate medical management.
PMCID: PMC3098047  PMID: 20837833
18.  Patient-specific induced pluripotent stem cell derived models of LEOPARD syndrome 
Nature  2010;465(7299):808-812.
Generation of reprogrammed induced pluripotent stem cells (iPSC) from patients with defined genetic disorders promises important avenues to understand the etiologies of complex diseases, and the development of novel therapeutic interventions. We have generated iPSC from patients with LEOPARD syndrome (LS; acronym of its main features: Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary valve stenosis, Abnormal genitalia, Retardation of growth and Deafness), an autosomal dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-MAPK signaling diseases, which also includes Noonan syndrome (NS), with pleiomorphic effects on several tissues and organ systems1,2. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSC have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LS-iPSC are larger, have a higher degree of sarcomeric organization and preferential localization of NFATc4 in the nucleus when compared to cardiomyocytes derived from human embryonic stem cells (HESC) or wild type (wt) iPSC derived from a healthy brother of one of the LS patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signaling pathways that may promote the disease phenotype.
PMCID: PMC2885001  PMID: 20535210
19.  Amniotic Fluid Cells Are More Efficiently Reprogrammed to Pluripotency Than Adult Cells 
Cellular Reprogramming  2010;12(2):117-125.
Recently, cultured human adult skin cells were reprogrammed to induced pluripotent stem (iPS) cells, which have characteristics similar to human embryonic stem (hES) cells. Patient-derived iPS cells offer genetic and immunologic advantages for cell and tissue replacement or engineering. The efficiency of generating human iPS cells has been very low; therefore an easily and efficiently reprogrammed cell type is highly desired. Here, we demonstrate that terminally differentiated human amniotic fluid (AF) skin cells provide an accessible source for efficiently generating abundant-induced pluripotent stem (AF-iPS) cells. By induction of pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4, and c-MYC) the terminally differentiated, cultured AF skin cells formed iPS colonies approximately twice as fast and yielded nearly a two-hundred percent increase in number, compared to cultured adult skin cells. AF-iPS cells were identical to hES cells for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity, in vitro and in vivo differentiation into the three germ layers and for their capacity to form embryoid bodies (EBs) and teratomas. Our findings provide a biological interesting conclusion that these fetal AF cells are more rapidly, easily, and efficiently reprogrammed to pluripotency than neonatal and adult cells. AF-iPS cells may have a “young,” more embryonic like epigenetic background, which may facilitate and accelerate pluripotency. The ability to efficiently and rapidly reprogram terminally differentiated AF skin cells and generate induced pluripotent stem cells provides an abundant iPS cell source for various basic studies and a potential for future patient-specific personalized therapies.
PMCID: PMC2998987  PMID: 20677926
20.  A large-scale survey of the novel 15q24 microdeletion syndrome in autism spectrum disorders identifies an atypical deletion that narrows the critical region 
Molecular Autism  2010;1:5.
The 15q24 microdeletion syndrome has been recently described as a recurrent, submicroscopic genomic imbalance found in individuals with intellectual disability, typical facial appearance, hypotonia, and digital and genital abnormalities. Gene dosage abnormalities, including copy number variations (CNVs), have been identified in a significant fraction of individuals with autism spectrum disorders (ASDs). In this study we surveyed two ASD cohorts for 15q24 abnormalities to assess the frequency of genomic imbalances in this interval.
We screened 173 unrelated subjects with ASD from the Central Valley of Costa Rica and 1336 subjects with ASD from 785 independent families registered with the Autism Genetic Resource Exchange (AGRE) for CNVs across 15q24 using oligonucleotide arrays. Rearrangements were confirmed by array comparative genomic hybridization and quantitative PCR.
Among the patients from Costa Rica, an atypical de novo deletion of 3.06 Mb in 15q23-q24.1 was detected in a boy with autism sharing many features with the other 13 subjects with the 15q24 microdeletion syndrome described to date. He exhibited intellectual disability, constant smiling, characteristic facial features (high anterior hairline, broad medial eyebrows, epicanthal folds, hypertelorism, full lower lip and protuberant, posteriorly rotated ears), single palmar crease, toe syndactyly and congenital nystagmus. The deletion breakpoints are atypical and lie outside previously characterized low copy repeats (69,838-72,897 Mb). Genotyping data revealed that the deletion had occurred in the paternal chromosome. Among the AGRE families, no large 15q24 deletions were observed.
From the current and previous studies, deletions in the 15q24 region represent rare causes of ASDs with an estimated frequency of 0.1 to 0.2% in individuals ascertained for ASDs, although the proportion might be higher in sporadic cases. These rates compare with a frequency of about 0.3% in patients ascertained for unexplained intellectual disability and congenital anomalies. This atypical deletion reduces the minimal interval for the syndrome from 1.75 Mb to 766 kb, implicating a reduced number of genes (15 versus 38). Sequencing of genes in the 15q24 interval in large ASD and intellectual disability samples may identify mutations of etiologic importance in the development of these disorders.
PMCID: PMC2907565  PMID: 20678247
21.  An atypical deletion of the Williams–Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism 
Journal of Medical Genetics  2006;44(2):136-143.
During a genetic study of autism, a female child who met diagnostic criteria for autism spectrum disorder, but also exhibited the cognitive–behavioural profile (CBP) associated with Williams–Beuren syndrome (WBS) was examined. The WBS CBP includes impaired visuospatial ability, an overly friendly personality, excessive non‐social anxiety and language delay.
Using array‐based comparative genomic hybridisation (aCGH), a deletion corresponding to BAC RP11‐89A20 in the distal end of the WBS deletion interval was detected. Hemizygosity was confirmed using fluorescence in situ hybridisation and fine mapping was performed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction.
The proximal breakpoint was mapped to intron 1 of GTF2IRD1 and the distal breakpoint lies 2.4–3.1 Mb towards the telomere. The subject was completely hemizygous for GTF2I, commonly deleted in carriers of the classic ∼1.5 Mb WBS deletion, and GTF2IRD2, deleted in carriers of the rare ∼1.84 Mb WBS deletion.
Hemizygosity of the GTF2 family of transcription factors is sufficient to produce many aspects of the WBS CBP, and particularly implicate the GTF2 transcription factors in the visuospatial construction deficit. Symptoms of autism in this case may be due to deletion of additional genes outside the typical WBS interval or remote effects on gene expression at other loci.
PMCID: PMC2598069  PMID: 16971481
22.  Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: Efficient identification of known microduplications and identification of a novel microduplication in ASMT 
BMC Medical Genomics  2008;1:50.
It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications.
In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region.
MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003).
MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.
PMCID: PMC2588447  PMID: 18925931

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