[Show abstract][Hide abstract] ABSTRACT: Two hereditary syndromes, lymphedema-distichiasis (LD) syndrome and blepharo-chelio-dontic (BCD) syndrome include the aberrant growth of eyelashes from the meibomian glands, known as distichiasis. LD is an autosomal dominant syndrome primarily characterized by distichiasis and the onset of lymphedema usually during puberty. Mutations in the forkhead transcription factor FOXC2 are the only known cause of LD. BCD syndrome consists of autosomal dominant abnormalities of the eyelid, lip, and teeth, and the etiology remains unknown. In this report, we describe a proband that presented with distichiasis, microcephaly, bilateral grade IV vesicoureteral reflux requiring ureteral re-implantation, mild intellectual impairment and apparent glomuvenous malformations (GVM). Distichiasis was present in three generations of the proband's maternal side of the family. The GVMs were severe in the proband, and maternal family members exhibited lower extremity varicosities of variable degree. A GLMN (glomulin) gene mutation was identified in the proband that accounts for the observed GVMs; no other family member could be tested. TIE2 sequencing revealed no mutations. In the proband, an additional submicroscopic 265 kb contiguous gene deletion was identified in 16q24.3, located 609 kb distal to the FOXC2 locus, which was inherited from the proband's mother. The deletion includes the C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci and 115 kb of a gene desert distal to FOXC2 and FOXL1. Thus, it is likely that the microcephaly, distichiasis, vesicoureteral, and intellectual impairment in this family may be caused by the deletion of one or more of these genes and/or deletion of distant cis-regulatory elements of FOXC2 expression.
American Journal of Medical Genetics Part A 04/2012; 158A(4):839-49. DOI:10.1002/ajmg.a.35229 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 4q deletion syndrome is a rare chromosome deletion syndrome with a wide range of clinical phenotypes. There is limited clinical phenotype and molecular correlation for congenital heart defects (CHDs) reported so far for this region primarily because many cases are large deletions, often terminal, and because high-resolution array has not been reported in the evaluation of this group of patients. CHDs are reported in about 60% of patients with 4q deletion syndrome, occurring in the presence or absence of dHAND deletion, implying the existence of additional genes in 4q whose dosage influences cardiac development. We report an 8-month-old patient with a large mid-muscular to outlet ventricular septal defect (VSD), moderate-sized secundum-type atrial septal defect (ASD), thickened, dysplastic pulmonary valve with mild stenosis and moderate pulmonic regurgitation, and patent ductus arteriosus (PDA). Illumina CytoSNP array analysis disclosed a de novo, heterozygous, interstitial deletion of 11.6 Mb of genomic material from the long arm of chromosome 4, at 4q32.3-q34.3 (Chr4:167236114-178816031; hg18). The deleted region affects 37 RefSeq genes (hg18), including two provisional microRNA stemloops. Three genes in this region, namely TLL1 (Tolloid-like-1), HPGD (15-hydroxyprostaglandin dehydrogenase), and HAND2 (Heart and neural crest derivatives-expressed protein 2), are known to be involved in cardiac morphogenesis. This report narrows the critical region responsible for CHDs seen in 4q deletion syndrome.
American Journal of Medical Genetics Part A 03/2012; 158A(3):635-40. DOI:10.1002/ajmg.a.34425 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Milroy disease, also known as primary congenital lymphedema, is a hereditary form of lymphedema with autosomal dominant inheritance. Individuals with Milroy disease are typically characterized by congenital onset of lymphedema of the lower limbs due to hypoplasia of the lymphatic vessels. The genetic basis of most cases of Milroy disease has not been established, although mutations in the vascular endothelial growth factor receptor VEGFR3 (FLT-4) are responsible for some cases with 17 mutations described to date. In this report, we describe a novel VEGFR3 mutation in exon 22 in a four-generation family in which congenital lymphedema segregates in an autosomal dominant manner. In addition to lymphedema, affected family members had other clinical manifestations associated with Milroy disease including hydrocele, ski jump toenails, large caliber veins, and subcutaneous thickening. We screened VEGFR3 for mutations which revealed a novel 3059A>T transversion in exon 22 resulting in Q1020L missense mutation in the second tyrosine kinase domain of VEGFR3. This mutant allele segregated with lymphedema among affected individuals with incomplete penetrance. This is the first report of an exon 22 mutation in Milroy disease.
American Journal of Medical Genetics Part A 06/2007; 143A(11):1212-7. DOI:10.1002/ajmg.a.31703 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GATA-2, a transcription factor that has been shown to play important roles in multiple organ systems during embryogenesis, has been ascribed the property of regulating the expression of numerous endothelium-specific genes. However, the transcriptional regulatory hierarchy governing Gata2 activation in endothelial cells has not been fully explored. Here, we document GATA-2 endothelial expression during embryogenesis by following GFP expression in Gata2-GFP knock-in embryos. Using founder transgenic analyses, we identified a Gata2 endothelium enhancer in the fourth intron and found that Gata2 regulation by this enhancer is restricted to the endocardial, lymphatic and vascular endothelium. Whereas disruption of three ETS-binding motifs within the enhancer diminished its activity, the ablation of its single E box extinguished endothelial enhancer-directed expression in transgenic mice. Development of the endothelium is known to require SCL (TAL1), and an SCL-E12 (SCL-Tcfe2a) heterodimer can bind the crucial E box in the enhancer in vitro. Thus, GATA-2 is expressed early in lymphatic, cardiac and blood vascular endothelial cells, and the pan-endothelium-specific expression of Gata2 is controlled by a discrete intronic enhancer.
Development 05/2007; 134(9):1703-12. DOI:10.1242/dev.001297 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular events involved in lymphatic development are poorly understood. Hence, the genes responsible for hereditary lymphedema are of great interest due to the potential for providing insights into the mechanisms of lymphatic development, the diagnosis, prevention and treatment of lymphedema, and lymphangiogenesis during tumor growth. Mutations in the FOXC2 transcription factor cause a major form of hereditary lymphedema, the lymphedema-distichiasis syndrome. We have conducted a study of Foxc2 expression during mouse development using immunohistochemistry, and examined its expression in lymphatics compared to its paralog Foxc1 and to Vegfr-3, Prox1 and other lymphatic and blood vascular proteins. We have found that Foxc2 is expressed in lymphatic primordia, jugular lymph sacs, lymphatic collectors and capillaries, as well as in podocytes, developing eyelids and other tissues associated with abnormalities in lymphedema-distichiasis syndrome.
[Show abstract][Hide abstract] ABSTRACT: To examine the FOXC2 gene in a family with hereditary distichiasis.
Distichiasis, ie, a second row of eyelashes arising from the meibomian glands of the eyelids, can be inherited either alone (Online Mendelian Inheritance in Man [OMIM] no. 126300) or, more commonly, as part of the lymphedema-distichiasis (LD) syndrome (OMIM no. 153400). More than 45 families with mutations in the FOXC2 gene and LD have been described. Both lymphedema and distichiasis are highly penetrant. Distichiasis without lymphedema is not commonly seen.
We examined three generations of a family (N = nine members) with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was polymerase chain reaction--amplified from genomic DNA from all family members and examined for mutations.
Clinical examination showed distichiasis of all four lids in two affected family members across two generations. There were no other consistent ophthalmologic abnormalities in the family. A cytosine-to-adenine transversion was identified in DNA from affected study participants at nucleotide position 1076, which would be predicted to cause truncation of the protein at codon 359. This change was not observed in any of the nine unaffected family members participating.
This finding suggests that hereditary distichiasis and LD may not be separate genetic disorders but different phenotypic expressions of the same underlying disorder. Ophthalmologists should be aware that LD may present as distichiasis alone and counsel and refer their patients appropriately.
Journal of American Association for Pediatric Ophthalmology and Strabismus 11/2003; 7(5):354-7. DOI:10.1016/S1091853103001447 · 1.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomic amplification can lead to the activation of cellular proto-oncogenes during tumorigenesis, and is observed in most, if not all, human malignancies, including adenocarcinomas of lung and esophagus. Using a two-dimensional restriction landmark genomic scanning technique, we identified five NotI/HinfI fragments with increased genomic dosage in an adenocarcinoma of the gastroesophageal junction. Four of these amplified fragments were matched within three contigs of chromosome 12 using the bioinformatics tool, Virtual Genome Scan. All three of the contigs map to the 12q13-q14 region, and the regional amplification in the tumor was verified using comparative genomic hybridization analysis. The 12q14 amplicon was characterized using sequence tagged site-amplification mapping with DNA from paired normal-tumor tissues of 75 gastroesophageal and 37 lung adenocarcinomas. The amplicon spans a region of >12 Mb between genes DGKA and BLOV1. The core-amplified domain was determined to be <0.5 Mb between marker WI-12457 and gene IFNG. However, MDM2, a well-documented oncogene of the region, is outside the core-domain. Eleven genes and expressed sequence tags within the amplicon were selected for quantitative reverse transcription-PCR, and DYRK2, a member of the dual-specificity kinase family, was overexpressed in all of the tumors showing gene amplification. Among the sequence tagged site/expressed sequence tag/gene markers tested, DYRK2 demonstrated the highest DNA copy number and the highest level of mRNA overexpression in the tumors. Moreover, DYRK2 mRNA overexpression (>2.5-fold of normal mean) was found in 18.6% of additional 86 lung adenocarcinomas in an assay using oligonucleotide microarrays. DYRK2 mRNA overexpression occurs more frequently than gene amplification in both esophageal and lung adenocarcinomas. This is the first report of amplification and overexpression of DYRK2 in any tumor type.
Cancer Research 07/2003; 63(14):4136-43. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lymphedema-distichiasis (LD) (OMIM 153400) is a rare autosomal-dominant condition characterized by pubertal onset of lower limb lymphedema and an aberrant second row of eyelashes arising from the meibomian glands. In some patients cardiac, skeletal and other defects coexist. We previously identified inactivating, nonsense and frameshift mutations in the forkhead transcription factor FOXC2 in affected members of LD families. To further delineate the relationship of FOXC2 deficiency to the clinical (and lymphangiodysplastic) phenotype in this syndrome, we performed dynamic lymphatic imaging and immunohistochemical examination of lymphatic tissues in mice heterozygous (+/-) for a targeted disruption of Foxc2. Adult heterozygote mice characteristically exhibited a generalized lymphatic vessel and lymph node hyper plasia and rarely exhibited hindlimb swelling. Retrograde lymph flow through apparently incompetent interlymphangion valves into the mesenteric nodes, intestinal wall and liver was also observed. In addition, Foxc2 +/- mice uniformly displayed distichiasis. We conclude that Foxc2 haploinsufficient mice mimic closely the distinctive lymphatic and ocular phenotype of LD patients. Furthermore, the craniofacial, cardiovascular and skeletal abnormalities sometimes associated with LD have previously been shown to be fully penetrant in homozygous Foxc2 null mice. This Foxc2 mutant mouse thus provides an ideal model for exploring molecular mechanisms and physiologic events in mesenchymal differentiation associated with lymphatic growth and development and the clinical abnormalities seen in human LD syndrome.
Human Molecular Genetics 06/2003; 12(10):1179-85. · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genomic amplification is observed in many, if not all, types of human malignancy and is one of the mechanisms for the activation of dominant-acting oncogenes in tumorigenesis. In the present study, three amplified restriction fragments were identified in an esophageal adenocarcinoma (P16) using the restriction landmark genome scanning two-dimensional gel technique. These fragments were cloned, sequenced, and mapped to chromosome band 14q13. Using the sequence tagged site-amplification mapping approach, we defined the core-amplified domain by screening 75 normal-tumor paired esophageal samples. The frequency of 14q13 amplification is 6.7% in esophageal tumors, and the amplicon spans >6 Mb in 1 tumor but is contained in a region <0.3 Mb in all of the remaining amplified tumors. Quantitative reverse transcription-PCR (RT-PCR) of 8 genes and expressed sequence tags located within the core-amplified domain revealed that the HNF3alpha (FOXA1)(4) gene, a forkhead gene family member, was overexpressed in all of the amplified esophageal tumors. HNF3alpha amplification was confirmed by Southern blot and interphase fluorescence in situ hybridization analyses, and the results of real-time RT-PCR were consistent with that of the regular quantitative RT-PCR. Increased immunohistochemical nuclear staining of the HNF3alpha protein was detected in all of the tumors containing 14q13 amplification. Affymetrix oligonucleotide microarrays of 86 lung adenocarcinomas demonstrated that expression of the HNF3alpha mRNA was elevated (> or =2.5-fold of mean expression in normal lung) in 37% (32 of 86) of the tumors analyzed. Gene amplification of HNF3alpha was detected in 2 of the 5 overexpressed lung tumors examined. This is the first report of HNF3alpha amplification, and overexpression in esophageal and lung adenocarcinomas. Amplification of HNF3alpha in esophageal and lung tumors may suggest a potential oncogenic role for this gene in tumorigenesis.
Cancer Research 09/2002; 62(18):5273-9. · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human ATOH7 gene encodes a basic helix-loop-helix (bHLH) transcription factor that is highly similar to Drosophila Atonal within the conserved bHLH domain. The ATOH7 coding region is contained within a single exon. We mapped ATOH7 to Chromosome (Chr) 10q21.3-22.1, a region syntenic to the segment of mouse Chr 10 where Atoh7 (formerly Math5) is located. The evolutionary relationship between ATOH7 and other atonal homologs was investigated using parsimony analysis. A direct comparison of ATH5/7 and ATH1 protein subgroups to Atonal also revealed a nonrandom distribution of amino acid changes across the bHLH domain, which may be related to their separate visual and proprioceptive sensory functions. Among bHLH genes, ATOH7 is most closely related to Atoh7. This sequence conservation extends significantly beyond the coding region. We define blocks of strong homology in flanking human and mouse genomic DNA, which are likely to include cis regulatory elements. Because targeted deletion of Atoh7 causes optic nerve agenesis in mice, we propose ATOH7 as a candidate for human optic nerve aplasia and related clinical syndromes.
[Show abstract][Hide abstract] ABSTRACT: Hereditary lymphoedema-distichiasis (LD) is an autosomal dominant disorder that classically presents as lymphoedema of the limbs, with variable age of onset, and extra aberrant growth of eyelashes from the Meibomian gland (distichiasis). Other major reported complications include cardiac defects, cleft palate, and extradural cysts. Photophobia, exotropia, ptosis, congenital ectropion, and congenital cataracts are additional eye findings. Recently, we reported that truncating mutations in the forkhead transcription family member FOXC2 resulted in LD in two families.
The clinical findings in seven additional families with LD, including the original family described by Falls and Kertesz, were determined and mutational analyses were performed.
Distichiasis was the most common clinical feature followed by age dependent lymphoedema. There is a wide variation of associated secondary features including tetralogy of Fallot and cleft palate. The mutational analyses identified truncating mutations in all of the families studied (two nonsense, one deletion, three insertion, and one insertion-deletion), which most likely result in haploinsufficiency of FOXC2.
FOXC2 mutations are highly penetrant with variable expressivity which is not explicable by the pattern of mutations.
Journal of Medical Genetics 12/2001; 38(11):761-6. · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Menkes disease and occipital horn syndrome (OHS) are allelic, X-linked recessive copper-deficiency disorders resulting from mutations in ATP7A, or MNK. Classic Menkes disease has a severe phenotype, with death in early childhood, whereas OHS has a milder phenotype, with, mainly, connective-tissue abnormalities. Data suggest that steady-state localization of ATP7A to the trans-Golgi network (TGN) is necessary for proper activity of lysyl oxidase, which is the predominant cuproenzyme whose activity is deficient in OHS and which is essential for maintenance of connective-tissue integrity. Recently, it was reported that ATP7A-transcript levels as low as 2%-5% of normal are sufficient to result in the milder phenotype, OHS, rather than the phenotype of Menkes disease. In contrast to previously reported cases of OHS, we describe a case of OHS in which, because of a frameshift mutation, no normal ATP7A is produced. Although abundant levels of mutant transcript are present, there are substantially reduced levels of the truncated protein, which lacks the key dileucine motif L1487L1488. It has been demonstrated that the dileucine motif L1487L1488 functions as an endocytic signal for ATP7A cycling between the TGN and the plasma membrane. The present report is the first to describe an ATP7A truncation that results in OHS rather than in Menkes disease. The data from the present report support the concepts that (1) OHS results from lower levels of functional ATP7A and (2) ATP7A does not require the dileucine motif to function in copper efflux.
The American Journal of Human Genetics 09/2001; 69(2):420-7. DOI:10.1086/321290 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Common fragile sites have been proposed to play a mechanistic role in chromosome translocations and other rearrangements in cancer cells in vivo based on their behavior in vitro and their co-localization with cancer translocation breakpoints. This hypothesis has been the subject of controversy, because associations have been made at the chromosomal level and because of the large number of both fragile sites and cancer chromosome breakpoints. Tests of this hypothesis at the molecular level are now possible with the cloning of common fragile site loci and the use of fragile site clones in the analysis of rearranged chromosomes. FRA3B, the most frequently seen common fragile site, lies within the large FHIT gene. It is now well established that this region is the site of frequent, large intragenic deletions and aberrant transcripts in a number of tumors and tumor cell lines. In contrast, only one tumor-associated translocation involving the FHIT gene has been reported. We have found translocations in both homologs of chromosome 3 in an early-passage esophageal adenocarcinoma cell line. This cell line showed no normal FHIT transcripts by reverse transcription polymerase chain reaction. Subsequent chromosome analysis showed translocations of the short arms of both homologs of chromosome 3: t(3;16) and t(3;4). The breakpoints of both translocations were shown by fluorescence in situ hybridization and polymerase chain reaction to be in the FHIT gene, at or near the center of the fragile site region. Using rapid amplification of cDNA ends with FHIT primers, a noncoding chimeric transcript resulting from t(3;16) was identified. These data provide direct support for the hypothesis that FRA3B, and likely other common fragile sites, may be "hot spots" for translocations in certain cancers, as they are for deletions, and that such translocations have the potential to form abnormal chimeric transcripts. In addition, the results suggest selection for loss of a functional FHIT gene by the translocation events.
Genes Chromosomes and Cancer 04/2001; 30(3):292-8. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Common fragile sites have been proposed to play a mechanistic role in chromosome translocations and other rearrangements in cancer cells in vivo based on their behavior in vitro and their co-localization with cancer translocation breakpoints. This hypothesis has been the subject of controversy, because associations have been made at the chromosomal level and because of the large number of both fragile sites and cancer chromosome breakpoints. Tests of this hypothesis at the molecular level are now possible with the cloning of common fragile site loci and the use of fragile site clones in the analysis of rearranged chromosomes. FRA3B, the most frequently seen common fragile site, lies within the large FHIT gene. It is now well established that this region is the site of frequent, large intragenic deletions and aberrant transcripts in a number of tumors and tumor cell lines. In contrast, only one tumor-associated translocation involving the FHIT gene has been reported. We have found translocations in both homologs of chromosome 3 in an early-passage esophageal adenocarcinoma cell line. This cell line showed no normal FHIT transcripts by reverse transcription polymerase chain reaction. Subsequent chromosome analysis showed translocations of the short arms of both homologs of chromosome 3: t(3;16) and t(3;4). The breakpoints of both translocations were shown by fluorescence in situ hybridization and polymerase chain reaction to be in the FHIT gene, at or near the center of the fragile site region. Using rapid amplification of cDNA ends with FHIT primers, a noncoding chimeric transcript resulting from t(3;16) was identified. These data provide direct support for the hypothesis that FRA3B , and likely other common fragile sites, may be ???hot spots??? for translocations in certain cancers, as they are for deletions, and that such translocations have the potential to form abnormal chimeric transcripts. In addition, the results suggest selection for loss of a functional FHIT gene by the translocation events. ?? 2001 Wiley-Liss, Inc.
Genes Chromosomes and Cancer 03/2001; 30(3):292-298. DOI:10.1002/1098-2264(2000)9999:9999<::AID-GCC1095>3.0.CO;2-F · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lymphedema-distichiasis (LD) is an autosomal dominant disorder that classically presents as lymphedema of the limbs, with variable age at onset, and double rows of eyelashes (distichiasis). Other complications may include cardiac defects, cleft palate, extradural cysts, and photophobia, suggesting a defect in a gene with pleiotrophic effects acting during development. We previously reported neonatal lymphedema, similar to that in Turner syndrome, associated with a t(Y;16)(q12;q24.3) translocation. A candidate gene was not found on the Y chromosome, and we directed our efforts toward the chromosome 16 breakpoint. Subsequently, a gene for LD was mapped, by linkage studies, to a 16-cM region at 16q24.3. By FISH, we determined that the translocation breakpoint was within this critical region and further narrowed the breakpoint to a 20-kb interval. Because the translocation did not appear to interrupt a gene, we considered candidate genes in the immediate region that might be inactivated by position effect. In two additional unrelated families with LD, we identified inactivating mutations-a nonsense mutation and a frameshift mutation-in the FOXC2 (MFH-1) gene. FOXC2 is a member of the forkhead/winged-helix family of transcription factors, whose members are involved in diverse developmental pathways. FOXC2 knockout mice display cardiovascular, craniofacial, and vertebral abnormalities similar to those seen in LD syndrome. Our findings show that FOXC2 haploinsufficiency results in LD. FOXC2 represents the second known gene to result in hereditary lymphedema, and LD is only the second hereditary disorder known to be caused by a mutation in a forkhead-family gene.
The American Journal of Human Genetics 01/2001; 67(6):1382-8. DOI:10.1086/316915 · 10.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A polymerase chain reaction (PCR)-based strategy was used to isolate a mouse cDNA (mCtr1) encoding a Cu transport protein. The deduced mCtr1 protein sequence exhibits 92% identity to human Ctr1, and has structural features in common with known high affinity Cu transporters from yeast. The expression of mouse Ctr1 functionally complements baker's yeast cells defective in high affinity Cu transport. Characterization of the mCtr1 genomic clone showed that the mCtr1 coding sequence is encompassed within four exons and that the mCtr1 locus maps to chromosome band 4C1-2. RNA blotting analysis demonstrated that mCtr1 is ubiquitously expressed, with high levels in liver and kidney, and early in embryonic development. Steady state mammalian Ctr1 mRNA levels were not changed in response to cellular Cu availability, which is distinct from the highly Cu-regulated transcription of genes encoding yeast high affinity Cu transporters. These studies provide fundamental information for further investigations on the function and regulation of Ctr1 in Cu acquisition in mammals.
[Show abstract][Hide abstract] ABSTRACT: The human FHIT gene is a putative tumor suppressor gene that maps to human chromosome band 3p14.2 in a region that is frequently deleted in cancers. It exhibits both genomic deletions and aberrant transcripts in a variety of tumors and spans the common fragile site FRA3B. This fragile site extends over a broad region of several hundred kb within the FHIT gene and may account for its instability in tumors. As one test of this hypothesis, we isolated the murine Fhit gene and asked whether it also contains a common fragile site and if it is unstable in mouse tumors or tumor cell lines. The Fhit gene was isolated, and the sequence was found to be 87.5% identical to that of the human FHIT gene in the open reading frame. Using fluorescence in situ hybridization, Fhit was assigned to mouse chromosome band 14A2, in a region that was previously shown to contain an aphidicolin-inducible mouse fragile site. Fluorescence in situ hybridization with genomic clones containing Fhit and flanking sequences demonstrated that gaps and breaks in the fragile site occur over a broad region within and proximal to the Fhit locus. Thus, the physical relationship of Fhit to a common fragile site is similar to that observed with the orthologous human FHIT gene and FRA3B.
Cancer Research 09/1998; 58(15):3409-14. · 9.33 Impact Factor