Sirenomelia and VACTERL association in the offspring of a woman with diabetes.

Sirenomelia and VACTERL association are defects of blastogenesis of unknown cause. Although they appear clinically distinct, some epidemiological and experimental studies suggest a common pathogenetic mechanism. We report on the reproductive history of a 28-year-old obese, diabetic mother who had three pregnancies. The first resulted in the birth of a sirenomelic child, the second in a miscarriage, while the third was terminated for fetal malformations, diagnosed post-mortem as VACTERL association. This observation supports the relationship between sirenomelia and VACTERL, which probably represent the two ends of the same phenotypic spectrum. Their occurrence in the same sibship also indicates a possible common cause. The coexistence with maternal diabetes seems more than a chance occurrence and the constellation of malformations observed in the present family may be explained as the pleiotropic effect of the same teratogenic agent interacting with genetic predisposition to diabetes and/or obesity.

A single nucleotide variant in the FMR1 CGG repeat results in a "Pseudodeletion" and is not associated with the fragile X syndrome phenotype.

The molecular diagnosis of fragile X syndrome relies on the detection of the pathogenic CGG repeat expansion in the FMR1 gene. Deletions and point mutations have occasionally been reported. Rare polymorphisms might mimic a deletion by Southern blot analysis, leading to false-positive results. We describe a novel rare nucleotide substitution within the CGG repeat. The proband was a woman with a positive family history of mental retardation. Southern blot analysis showed an additional band consistent with a deletion in the region detected by the StB12.3 probe. Sequencing of this region revealed a G>C transversion that interrupts the CGG repeat and introduces an EagI site. The same variant was observed in both the healthy son and father of the proband, supporting the hypothesis that the nucleotide substitution is a silent polymorphism, the frequency of which we estimated to be less than 1% in the general population. These findings argue for a pathogenic role of nucleotide variants within the CGG repeat and suggest possible consequences of unexpected findings in the molecular diagnostics of fragile X syndrome. Thus, although the sequence context of a single nucleotide substitution may not predict possible effects on mRNA or protein function, a specific change in the higher order structures of DNA or mRNA may be functionally relevant in the pathological phenotype.

Tibial developmental field defect is the most common lower limb malformation pattern in VACTERL association.

VACTERL association is one of the most common recognizable patterns of human malformation and has been recently defined as a multiple polytopic developmental field defect. Limb anomalies are a key component of this condition and characteristically reflect perturbation of radial ray development. However, the pattern of appendicular malformations in VACTERL association is wider and includes a broad spectrum of additional and apparently nonspecific anomalies. We report on the sporadic case of a 4-10/12-year-old boy presenting with multiple costovertebral defects, dextrocardia, bilateral radial ray hypo/aplasia, unilateral kidney agenesis and anal atresia. Homolaterally to the more severe radial ray defect and kidney aplasia, he also has a complex lower limb malformation, consisting of distal tibial aplasia, clubfoot, hallucal deficiency and preaxial polydactyly. Literature review identifies 24 additional patients with VACTERL manifestations and lower limb malformations (excluding cases with isolated secondary deformations). Tibial hypo/aplasia with or without additional tibial field defects, reported in about 2/3 (68%) of the patients, represents the most common finding, while involvement of the fibular ray is rare (20%) and very often accompanies tibial anomalies. The relatively high frequency of tibial ray anomalies in VACTERL patients could easily be explained by the principle of homology of the developmental field theory. Careful search of lower limb anomalies of the "tibial type" is, therefore, indicated in all patients with multiple polytopic developmental field defects.

The antigen processing machinery of class I human leukocyte antigens: linked patterns of gene expression in neoplastic cells.

The ultimate outcome of an immune response (escape or surveillance) depends on a delicate balance of opposing signals delivered by activating and inhibitory immune receptors expressed by cytotoxic T lymphocytes and natural killer cells. In this light, loss and down-regulation of human leukocyte antigens (HLA) class I molecules, while important for keeping tumors below the T-cell detection levels, may incite recognition of missing self. Conversely, the maintenance of normal levels of expression (or even up-regulation) may be favorable to tumors, at least in certain cases. In this study, we took advantage of a previously characterized panel of 15 early passage tumor cell lines (mainly from melanoma and lung carcinoma lesions) enriched with class I-low phenotypes. These cells were systematically characterized by Northern and/or Western blotting (e.g., mini-transcriptome/mini-proteome analysis) for the expression of HLA-A, -B, -C, beta(2)-microglobulin, and the members of the "antigen processing machinery" of class I molecules (LMP2, LMP7, TAP1, TAP2, tapasin, calreticulin, calnexin, and ERp57). In addition, we established four pairs of cultures, each comprising melanoma cells and normal melanocytes from the same patient. We found that approximately 97% of the 185 tested gene products are expressed (although often weakly), and in many cases coordinately regulated in 18 of 19 tumor cell lines. Linked expression patterns could be hierarchically arranged by statistical methods and graphically described as a class I HLA "coordinome." Deviations (both down- and up-regulation) from the coordinome expression pattern inherited from the normal, paired melanocyte counterpart, were allowed but limited in magnitude, as if melanoma cells were trying to keep a "low profile" HLA phenotype. We conclude that irreversible HLA loss is a rare event, and class I expression in tumor cells almost invariably results from reversible gene regulatory (rather than gene disruption) events.

A melanoma immune response signature including Human Leukocyte Antigen-E.

Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E (HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis.

cDNA-array profiling of melanomas and paired melanocyte cultures.

Three paired (from the same donor) sets of melanoma cells and normal melanocytes, established as early-passage cultures from metastatic lesions and the uninvolved skin of three patients, were comparatively cDNA profiled by macroarrays (approximately 1,200 genes) and reverse transcription (RT)-PCR. While 145 gene products were significantly (at least twofold) upregulated or downregulated in at least 1 pair, and 23 were in at least 2 pairs, only 3 (the signal transducer and activator of transcription STAT2, collagen type VI, and CD9) were concordantly modulated (downregulation) in all 3 pairs. Array results were validated by RT-PCR on a small panel of surgically removed nevocellular nevi and metastatic melanoma lesions, and by immunohistochemistry on a large panel of benign and malignant lesions of the nevomelanocytic lineage. The three gene products were downregulated at different stages of melanoma progression. STAT2 was detectable in nevi (5/5) and most primary melanomas (11/12), but was lost in 10/15 metastatic lesions. Collagen type VI was expressed in nevi (5/5) and primary melanomas below a Breslow thickness of 1 mm (3/3), but was lost in 24/24 primary melanomas above this threshold, and in metastatic melanomas (10/10). The tetraspanin CD9 molecule was expressed in 18/18 nevi, but was lost in 20/28 primary melanomas (including thin lesions), and in 24/52 metastatic lesions. These data provide the proof of principle that cDNA profiling of paired melanocyte/melanoma cultures sorts out novel, early signatures of melanocyte transformation that could contribute to the clinical management of patients at high risk of metastatic disease.