Bethlem myopathy: long-term follow-up identifies COL6 mutations predicting severe clinical evolution.

OBJECTIVE: Mutations in one of the 3 genes encoding collagen VI (COLVI) are responsible for a group of heterogeneous phenotypes of which Bethlem myopathy (BM) represents the milder end of the spectrum. Genotype-phenotype correlations and long-term follow-up description in BM remain scarce. METHODS: We retrospectively evaluated the long-term clinical evolution, and genotype-phenotype correlations in 35 genetically identified BM patients (23 index cases). RESULTS: Nineteen patients showed a typical clinical picture with contractures, proximal weakness and slow disease progression while 11 presented a more severe evolution. Five patients showed an atypical presentation, namely a limb girdle muscle weakness in 2 and a congenital myopathy pattern with either no contractures, or only limited to ankles, in 3 of them. Pathogenic COL6A1-3 mutations were mostly missense or in frame exon-skipping resulting in substitutions or deletions. Twenty one different mutations were identified including 12 novel ones. The mode of inheritance was, autosomal dominant in 83% of the index patients (including 17% (N=4) with a de novo mutation), recessive in 13%, and undetermined in one patient. Skipping of exon 14 of COL6A1 was found in 35% of index cases and was mostly associated with a severe clinical evolution. Missense mutations were detected in 39% of index cases and associated with milder forms of the disease. CONCLUSIONS: Long-term follow-up identified important phenotypic variability in this cohort of 35 BM patients. However, worsening of the functional disability appeared typically after the age of 40 in 47% of our patients, and was frequently associated with COL6A1 exon 14 skipping.

Mutations in a polycistronic nuclear gene associated with molybdenum cofactor deficiency.

All molybdoenzymes other than nitrogenase require molybdopterin as a metal-binding cofactor. Several genes necessary for the synthesis of the molybdenum cofactor (MoCo) have been characterized in bacteria and plants. The proteins encoded by the Escherichia coli genes moaA and moaC catalyse the first steps in MoCo synthesis. The human homologues of these genes are therefore candidate genes for molybdenum cofactor deficiency, a rare and fatal disease. Using oligonucleotides complementary to a conserved region in the moaA gene, we have isolated a human cDNA derived from liver mRNA. This transcript contains an open reading frame (ORF) encoding the human moaA homologue and a second ORF encoding a human moaC homologue. Mutations can be found in the majority of MoCo-deficient patients that confirm the functional role of both ORFs in the corresponding gene MOCS1 (for 'molybdenum cofactor synthesis-step 1'). Northern-blot analysis detected only full-length transcripts containing both consecutive ORFs in various human tissues. The mRNA structure suggests a translation reinitiation mechanism for the second ORF. These data indicate the existence of a eukaryotic mRNA, which as a single and uniform transcript guides the synthesis of two different enzymatic polypeptides with disease-causing potential.

Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors.

In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells.

Very long chain acyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid oxidation in fibroblasts.

A patient highly suspected of long chain fatty acid oxidation defect was investigated for membrane-bound palmitoyl-CoA dehydrogenation in a membrane extract from skin fibroblasts, using 1% sodium cholate as detergent. The profoundly decreased activity observed is consistent with a deficiency of the newly identified mitochondrial 'very long chain acyl-CoA dehydrogenase'.

Culture of putative Langerhans cell bone marrow precursors: characterization of their phenotype.

We searched for the presence of human CD1-positive cells in bone marrow populations in order to characterize putative Langerhans cell precursors. Bone marrow progenitors were cultured in 0.8% methylcellulose supplemented with 10% granulocyte-macrophage (GM) colony-stimulating factor(s) GCT and HTB9. We compared the kinetics of these two factors and found that GCT was the more appropriate for our study. After 8 days of culture, colony-forming units of granulocyte-macrophages (CFU-GM) were tested for the presence of CD1-positive cells using the immunofluorescence technique. Positive cells were counted by cytofluorometric analysis: 9.4% CD1a (BL6), 13.4% CD1c (L161), 4.3% CD1b (NuT2), 4.6% CD2 (T11), and 25.5% CD33 (My9). Ultrastructural features and phenotype were then specified by the immunogold labeling technique using electron microscopy. A subpopulation of CD1-positive cells showed the ultrastructural morphology of bone marrow pro-monocyte/monocyte cells. By using well-characterized monoclonal antibodies, it was demonstrated that these cells expressed the following phenotype: CD14+, CD33+, CD4+, HLA-DR+, HLA-DP+, HLA-DQ-, OKT10-, CD2-. These data indicate that these bone marrow promonocyte/monocyte progenitors express a phenotype similar to that of epidermal Langerhans cells but the density of each antigen is much lower than that observed on mature skin dendritic cells.

Fifteen new mutations (-195C>T, L-12X, 298-2A>G, T117N, A159T, R229S, 997+2T>A, E274X, A331T, H364R, D389G, 1256delC, R433H, N461I, C472S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and liver/bone/kidney-type alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 12 families affected by severe or mild hypophosphatasia. Twenty distinct mutations were found, 5 of which were previously reported. Nine of the 15 new mutations were missense mutations (T117N, A159T, R229S, A331T, H364R, D389G, R433H, N461I, and C472S). The others were 2 nonsense mutations (L-12X and E274X), one single nucleotide deletion (1256delC), 2 mutations affecting splicing (298-2A>G, 997+2T>A), and a mutation in the major transcription start site (-195C>T). Hum Mutat 15:293, 2000.

Concentration of 14 steroid hormones in human amniotic fluid of midpregnancy.

Amniotic fluid (AF) levels of all steroids leading from pregnenolone (delta 5Preg) to androgens and estrogens of both the delta 5 and the delta 4 pathways and those of cortisol and cortisone have been determined in 63 normal pregnancies (12-19 weeks gestation). The 12 unconjugated steroids [delta 5Preg, 17 alpha-hydroxypregnenolone, progesterone, 17 alpha-hydroxyprogesterone, dehydroepiandrosterone (DHA), delta 4-androstenedione, delta 5-androstene-3 beta,17 beta-diol (delta 5Adiol), testosterone (T), estrone, 17 beta-estradiol (E2), cortisol, and cortisone] were measured by specific RIAs after appropriate purification by Celite or LH-20 column chromatography, while the sulfates of DHA and delta 5Preg were assayed directly on diluted samples. There were distinct sex differences; T and delta 4-androstenedione levels were higher (P < 0.001) in males than in females, while AF levels of delta 5Preg, 17 alpha-hydroxypregnenolone, 17-hydroxyprogesterone, DHA, and delta 5Adiol were higher (P < 0.05) in females than in males. AF levels of E2 were significantly higher in females only between 15 and 19 weeks gestation. There was no difference between sexes in AF levels of progesterone, estrone, cortisone, and cortisol. AF levels of T, delta 4-androstenedione, and E2 decreased with age in males, and AF levels of DHA increased in females during the period examined. These observed sex differences suggest that T and delta 4-androstenedione may reflect fetal testicular activity, while E2 and 17-hydroxyprogesterone might reflect fetal ovarian activity. Determination of AF levels of T would appear to be a valuable screening test for antenatal diagnosis of sex (predictive error, less than or equal to 15%), but not in the presence of steroidogenic enzyme defects. Elevated levels of 17-hydroxyprogesterone were found in the AF of two fetuses with either a 17-20 desmolase defect or 21-hydroxylase deficiency; AF levels of androgens were low in the former and high in the latter.

Pathogenicity of missense mutations in SURF1 deficiency inducing the Leigh syndrome. Importance in diagnosis.

Leigh syndrome with cytochrome oxidase (COX) deficiency has been associated with SURF1 mutations. For patient diagnosis, distinction between neutral polymorphisms and pathogenic missense SURF1 mutations in Leigh syndrome is essential. We show that several missense SURF1 mutations did not allow a stable protein to be expressed. Absence of immunologically reactive SURF1 is, therefore, helpful to demonstrate their pathogenicity. In addition, we show that out of two previously described missense mutations housed by the same allele, only one, the T737 C was pathogenic. Indeed, transfection of T737 C mutated SURF1 in SURF1-deficient cells did not restore normal SURF1 stability and COX activity. On the contrary, the G604 C-mutated SURF1 did it and, hence, is a neutral variant.

Fetal liver transplantation: biology and clinical results.

Over the last 18 years, we have developed the transplantation of fetal liver cells to treat severe immunodeficiencies, hematological disorders and inborn errors of metabolism. Post-natally, this treatment is successful in two-third of patients and it is therefore very valuable, especially when there is no perfectly matched donor for a bone marrow transplant. Since 1988 we have carried out these fetal liver transplants (FLTs) in utero, immediately after prenatal diagnosis. Engraftment and reconstitution have been obtained, and several advantages appear to be associated with in utero FLT: increased probability of graft take, ideal isolation of the patient (in the maternal uterus) and optimal environment for the differentiation of the transplanted fetal liver cells (in the fetal host).

In utero transplantation of stem cells in humans: immunological aspects and clinical follow-up of patients.

Four human fetuses were treated by transplantation of human fetal liver stem cells. Two of them had severe immunodeficiency disease and the two other ones had thalassemia major. Three of these in utero transplants were followed by engraftment. The three patients are now born: the first one is now very healthy thanks to the reconstitution of cell-mediated immunity associated with this transplant, and he lives normally at home; the two other ones, who have been more recently treated, have a significant improvement of their condition and they also live normally at home. This procedure, for the first time used in humans, has therefore demonstrated its feasibility and its efficacy: during early fetal development, foreign cells engraft readily and may result in cure or significant correction of a large variety of inherited diseases.

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency: clinical characteristics and diagnostic considerations in 30 patients.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an enzyme catalyzing the dehydrogenation of long-chain fatty acids in the first step of mitochondrial fatty acid oxidation. Using an ETF (electron transfer flavoprotein, the physiological electron acceptor of VLCAD) reduction assay, we identified VLCAD deficiency in cultured skin fibroblasts or liver tissue from 30 patients in 27 families. They clinically presented two phenotypes: a 'severe' presentation characterized by an early onset of symptoms, with hypertrophic cardiomyopathy and a high incidence of death, and a 'mild' form with hypoketotic hypoglycaemia, resembling MCAD (medium-chain acyl-CoA dehydrogenase) deficiency. Cells isolated from patients who develop cardiomyopathy characteristically accumulate longer-chain length acylcarnitines (hexadecanoylcarnitine and tetradecanoylcarnitine) when incubated with palmitate. However, cells from patients with the hypoglycaemic presentation produced relatively shorter-chain-length intermediates (mainly dodecanoylcarnitine). Inhibition of carnitine palmitoyl transferase I, in vitro, eliminated these intermediates with cells from both phenotypes indicating their intramitochondrial origin. Although the explanation for these distinct biochemical findings is not obvious, the correlation with the two phenotypes provides an opportunity for accurate prognosis and early implementation of appropriate treatment. Prenatal diagnosis of this life-threatening disorder was successfully performed in seven pregnancies in six of those families by assay of trophoblasts or amniocytes. In an at risk family, diagnosis of an affected fetus by measurement of VLCAD activity in noncultured chorionic villi allowed termination of the pregnancy before 13 weeks of gestation.

Oxidation of unsaturated fatty acids by human fibroblasts with very-long-chain acyl-CoA dehydrogenase deficiency: aspects of substrate specificity and correlation with clinical phenotype.

The degradation of unsaturated fatty acids was examined in fibroblasts from 16 patients with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. Analysis of acylcarnitine intermediates following incubation of intact human cells with these compounds revealed that the milder clinical phenotypes could be distinguished from the severe cardiomyopathic phenotype. These findings may reflect more effective contributions of alternate pathways in the milder forms of the disease. Incubation of VLCAD-deficient cells with cis-9 or trans-9 unsaturated fatty acids indicate that VLCAD is largely responsible for the 2,3-dehydrogenation of cis-5 or trans-5 intermediates in fibroblasts. The first two cycles of beta-oxidation with oleic and linoleic acids occur in the absence of VLCAD activity suggesting the presence of an additional acyl-CoA dehydrogenase or alternate pathway for the oxidation of these unsaturated fatty acids. These observations have clinical relevance for determining diagnosis, prognosis and strategies for dietary treatment of these patients.

[Spontaneous colonic perforations revealing Ehlers-Danlos syndrome type IV]. / Perforations coliques spontanées révélatrices d'un syndrome d'Ehlers-Danlos de type IV.

BACKGROUND: The malignant form of Ehlers-Danlos syndrome type IV owes its bad reputation to a proneness to spontaneous rupture of bowel or large vessels, which may reveal the disease. CASE REPORT: A girl suffered acute rupture of the sigmoid at the age of 5 years and rupture of the left colon, twice, at the age of 11 and 13 years, respectively. These ruptures required colostomy and finally colectomy. A proneness to bruisability, history of dislocation of hips, hypermobile joints, ovarian cysts and some minor abnormalities of her face resembled that of the Ehlers-Danlos syndrome which was confirmed by optic and electronic microscopy of the skin biopsy. CONCLUSION: This is the youngest case of rupture of bowel reported in Ehlers-Danlos syndrome. Long-term prognosis is influenced by repetition of intestinal ruptures and occurrence of vascular complications.

[Not Available].

Synopsis The normal dermal human fibroblastic cell (NDHF) was used to determine a cellular ageing pattern. Cells were cultured in monolayers until the 30th passage. First of all, the following cell growth characteristics were studied: growth rate, fluorimetric DNA determination, DNA repair after UV irradiation. Secondly, metabolism characteristics were examined: lysosomal enzymatic activity and type I and III collagen biosynthesis. Strains were obtained from 10,30,43 and 69-year-old donors to favour a comparison between in vitro and in vivo ageing. Cell growth ability is modified in vitro only for the oldest strain which shows a significant decrease in the cellular density at the 30th passage. The DNA rate and its repairing ability are not changed by in vitro ageing whatever the strain age. Lysosomal activity increases during in vitro ageing whereas the collagen I synthesis decreases. In vitro proliferating potentialities do not reflect in vivo ageing. On the other hand, in this study, metabolic potentialities evolve in the same way in vitro as in vivo and could be a good enough pattern to select anti-ageing products.

X-linked recessive ichthyosis. Enzymatic diagnosis of affected males and female carriers.

Steroid sulfatase deficiency (SSD) is a sex-linked disorder characterized clinically by generalized X-linked ichthyosis. We report a study of 10 families where the clinical diagnosis of this disorder was confirmed by measuring arylsulfatase C and steroid sulfatase (STS) in cultured skin fibroblasts and/or leukocytes of patients and heterozygotes. The optimal conditions for these enzymatic determinations were determined. Our data indicate that STS measurement is a reliable test for SSD diagnosis, either in fibroblasts or in leukocytes. For the detection of heterozygotes, several enzymatic determinations in different cell types are required.