Clinical heterogeneity in lymphoedema-distichiasis with FOXC2 truncating mutations.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: FOXC2 mutations are highly penetrant with variable expressivity which is not explicable by the pattern of mutations.

A novel frameshift mutation in exon 23 of ATP7A (MNK) results in occipital horn syndrome and not in Menkes disease.

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.

Translocation breakpoints in FHIT and FRA3B in both homologs of chromosome 3 in an esophageal adenocarcinoma.

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.

Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome.

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.

Isolation of a murine copper transporter gene, tissue specific expression and functional complementation of a yeast copper transport mutant.

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.

The murine Fhit gene is highly similar to its human orthologue and maps to a common fragile site region.

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.