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Background: T cell-derived opioids play a key role in the control of inflammatory pain. However, the nature of opioids produced by T cells is still matter of debate in mice. Whereas β-endorphin has been found in T lymphocytes by using antibody-based methods, messenger RNA (mRNA) quantification shows mainly mRNA encoding for enkephalins. The objective of the study is to elucidate the nature of T cell-derived opioids responsible for analgesia and clarify discrepancy of the results at the protein and genetic levels., Methods: CD4(+) T lymphocytes were isolated from wild-type and enkephalin-deficient mice. mRNA encoding for β-endorphin and enkephalin was quantified by RT-qPCR. The binding of commercially available polyclonal anti-endorphin antibodies to lymphocytes from wild-type or enkephalin knockout mice was assessed by cytofluorometry. Opioid-mediated analgesic properties of T lymphocytes from wild-type and enkephalin-deficient mice were compared in a model of inflammation-induced somatic pain by measuring sensitivity to mechanical stimuli using calibrated von Frey filaments., Results: CD4(+) T lymphocytes expressed high level of mRNA encoding for enkephalins but not for β-endorphin in mice. Anti-β-endorphin polyclonal IgG antibodies are specific for β-endorphin but cross-react with enkephalins. Anti-β-endorphin polyclonal antibodies bound to wild-type but not enkephalin-deficient CD4(+) T lymphocytes. Endogenous regulation of inflammatory pain by wild-type T lymphocytes was completely abolished when T lymphocytes were deficient in enkephalins. Pain behavior of immune-deficient (i.e., without B and T lymphocytes) mice was superimposable to that of mice transferred with enkephalin-deficient lymphocytes., Conclusions: Rabbit polyclonal anti-β-endorphin serum IgG bind to CD4(+) T lymphocytes because of their cross-reactivity towards enkephalins. Thus, staining of T lymphocytes by anti-β-endorphin polyclonal IgG reported in most of studies in mice is because of their binding to enkephalins. In mice, CD4(+) T lymphocytes completely lose their analgesic opioid-mediated activity when lacking enkephalins.

Background & Aims: A dysregulated response of CD4(+) T cells against the microbiota contributes to the development of inflammatory bowel disease. Effector CD4(+) T cells, generated in response to microbe-derived antigens, can reduce somatic inflammatory pain through the local release of opioids. We investigated whether colitogenic CD4(+) T cells that accumulate in the inflamed colon also produce opioids and are able to counteract inflammation-induced visceral pain in mice., Methods: Colitis was induced via transfer of naive CD4(+)CD45RB(high) T cells to immune-deficient mice or by administration of dextran sulfate sodium. Mice without colitis were used as controls. Samples of colon tissue were collected, and production of opioids by immune cells from inflamed intestine was assessed by quantitative polymerase chain reaction and cytofluorometry analyses. The role of intestinal opioid tone in inflammation-induced visceral hypersensitivity was assessed by colorectal distention., Results: In mice with T cell- or dextran sulfate sodium-induced colitis, colitogenic CD4(+) T cells (T-helper 1 and Th17 cells) accumulated in the inflamed intestine and expressed a high level of endogenous opioids. In contrast, macrophages and epithelial cells did not express opioids; opioid synthesis in the myenteric plexus was not altered on induction of inflammation. In mice with colitis, the local release of opioids by colitogenic CD4(+) T cells led to significant reduction of inflammation-associated visceral hypersensitivity., Conclusions: In mice, colitogenic Th1 and Th17 cells promote intestinal inflammation and colonic tissue damage but have simultaneous opioid-mediated analgesic activity, thereby reducing abdominal pain., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

Endogenous opioid peptides mainly produced by neurons are also released by immune cells. They bind to mu- (mu-opioid receptor, MOR), delta-, and kappa-opioid receptors. On the basis of studies on mice showing that MOR is the main mediator of the deleterious effects of opioids on immunity, we wondered whether MOR, absent under normal conditions, is expressed in some pathological situations such as lymphomas. mRNA expression for all three opioid receptors was examined in lymph node biopsy samples from patients with non-Hodgkin's B-cell and T-cell lymphomas. We found that MOR and one of its ligands (enkephalin) are simultaneously expressed almost exclusively in lymph nodes from patients with Sézary cutaneous T cell lymphoma. As MOR was undetectable in circulating malignant T lymphocytes and in normal immune cells, we hypothesized that tumor-released cytokines might induce MOR expression in non-neoplastic lymph node cells. The correlation between mRNA levels of MOR and interleukin-13 (IL-13) within lymph nodes from Sézary patients led us to investigate the ability of IL-13 to upregulate MOR expression in normal immune cell subsets. We found that IL-13 upregulates MOR in activated Langerhans cells. Thus, our data suggest that, under pathological conditions, IL-13 overexpression might allow immune-derived endogenous opioids to down-modulate immune response.

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

A large family (MRX48) with a nonspecific X-linked mental retardation condition is described. An X-linked semidominant inheritance is suggested by the segregation in three generations of a moderate to severe mental retardation in seven males and by a milder intellectual impairment in two females, without any specific clinical, radiological, or biological feature. Two-point linkage analysis demonstrated significant linkage between the disorder and several markers in Xq28 (maximum LOD score [Zmax] = 2.71 at recombination fraction [theta] = 0); multipoint linkage analyses confirmed the significant linkage with a Zmax of 3.3 at theta = 0, at DXS1684. A recombination event observed with the flanking marker DXS8011 delineates a locus between this marker and the telomere. The approximate length of this locus is 8-9 cM, corresponding to 5.5-6 Mb. In an attempt to explain the variable intellectual impairment in females, we examined X-chromosome inactivation in all females of the family. Inactivation patterns in lymphocytes were random or moderately skewed, and no correlation between the phenotypic status and a specific inactivation pattern was observed. The interval of assignment noted in this family overlaps with five MRX loci previously reported in Xq28.

Pallister-Killian syndrome is a clinically recognizable syndrome characterized by tissue-limited mosaicism for an extra 12p isochromosome. Very little is known about the underlying mechanism of this rare rearrangement. Microsatellite markers were studied from three fetuses with Pallister-Killian syndrome and their parents to determine the parent of origin and the cell division yielding the additional isochromosome. In two cases the isochromosome contained the same allele(s) as a normal transmitted chromosome 12, one paternal and one maternal in origin. A third case showed inheritance of two different maternal alleles, indicating that at least one meiotic error was involved in the ultimate formation of the extra isochromosome.

In fragile X syndrome, the most common cause of inherited mental retardation, phenotypic expression has been linked to a region containing a repetitive sequence, (CGG)n, that appears to lengthen dramatically in fragile X patients and to show length variation in normal individuals. In order to investigate possible mechanisms responsible for further expansion of CGG in the normal population, we selected 31 normal unrelated X chromosomes carrying either the high-risk DX204-AC155 or DX196-AC151 haplotypes, as defined by the flanking microsatellites, DXS548 and FRAXAC2. Nearly 100% of CGGs with more than 35 repeats were found on DX204-AC155 haplotypes, with a mean length significantly higher and much more variable than in normal individuals carrying other haplotypes including the high-risk haplotype DX196-AC151. These findings suggest that the transition from the normal to the abnormal range occurs by a multistep process, a primary event, such as unequal crossing-over, leading to increased size and moderate instability of the repeat, and from which DNA polymerase slippage could lead to recurrent premutations. Our results also suggest that the upper limit of the normal range is roughly 35 repeats in the fragile X gene. The 36-54 repeats range would define an intermediate allele only observed, up to now, in DX204-AC155 fragile X chromosomes.

The phenotypically normal sister of a patient affected by fragile X syndrome was referred for genetic counselling and was found to carry a mosaic karyotype 46,X,r(X)/45,X. Because the probability of the simultaneous chance occurrence of fragile X syndrome and a ring chromosome X in the same family is very low, we postulated that the breakpoint of the ring chromosome X originated in the cytogenetic break in Xq27.3 responsible for fragile X syndrome. In order to determine the relative positions of the breakpoint on the ring chromosome X and the (CGG)n unstable sequence responsible for the fragile X mutation, we used molecular markers to analyse the telomeric regions of chromosome X in this family. The results showed that the ring chromosome X was the maternal fragile X chromosome and that the telomeric deletion on the long arm encompassed the (CGG)n sequence. This suggests that the cytogenetic break in Xq27.3 is distinct from the unstable (CGG)n sequence, or that the break followed by the end-to-end fusion creating the ring chromosome was not completely conservative. Analysis of DNA markers on the short arm of chromosome X evidenced a deletion of a large part of the pseudoautosomal region, allowing us to position the genes involved in stature and in some syndromes associated with telomeric deletions of Xp on the proximal side of the pseudoautosomal region.

The sizes of the fragile X mutation in 33 sib pairs affected with fragile X syndrome were determined by Southern blot analysis. An age-dependent decrease in the size of the mutation was found, suggesting positive selection of blood cells carrying small mutations during life or maternal imprinting.

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X-linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.

Some 250 different mutations have so far been screened in the cystic fibrosis (CF) gene. The 50 nonsense, 33 splicing and 60 frameshift mutations are randomly distributed within the gene, unlike the 107 missense mutations or amino acid deletions. A large excess of missense mutations affects the exons encoding the first transmembrane (MS1) and first ATP-binding fold (NBF1) domains. Sixty-four of the 107 missense mutations may be classified as private, demic, local and general mutations on the basis of their geographic distribution in Europe. Private and demic mutations are randomly distributed within the gene; local and general mutations are not. It is well known that some RFLP markers are in linkage disequilibrium with some mutations. Private, demic and local mutations are randomly associated with each class of RFLP haplotypes. In contrast, general mutations, frequent and infrequent, are not randomly associated with RFLP markers. General mutations usually affect a specific part of the gene and are more likely to be associated with a specific RFLP marker. This suggests the existence of selective factors favoring these mutations, a hypothesis formerly postulated as a possible cause of the high frequency of the disease.

Thirty-seven pregnancies at risk for Niemann-Pick type C disease were monitored by study of cultured amniotic fluid cells (8 cases) or chorionic villus cells (29 cases) in 23 couples over the period 1984-91. An early protocol combined determination of sphingomyelinase activity with electron microscopy. The current strategy, based on the demonstration of specific abnormalities in intracellular processing of exogenous cholesterol, combines the study of the early phase (first 6 h) of LDL-induced cholesteryl ester formation and the histochemical evaluation (filipin staining after 24 h of LDL uptake) of the LDL-induced accumulation of unesterified cholesterol. Thirteen fetuses were predicted to be affected. Confirmation of the diagnosis was made by study of cholesterol processing in fetal skin fibroblast cultures and/or by demonstration of a characteristic lipid storage in fetal liver, already present at 14 w gestation. Definition of the biochemical phenotype (classical, variant, or intermediate) of the index case, with regard to cholesterol-processing abnormalities, is an absolute prerequisite to adequate genetic counseling in a given family. Prenatal diagnosis has now proved a safe procedure in the predominant (approximately 85%) group of families with the classical phenotype.

A technique allowing the simultaneous screening of the four main CF mutations in the French population (delta F508, delta I507, G542X, S549N) has been developed by means of allele sequence-specific oligonucleotide (ASO) reverse dot blot. Using a strategy proposed by R. K. Saïki et al. (1989, Proc. Natl. Acad. Sci. USA 86: 6230-6234) for HLA-DQA, the seven ASOs for normal and mutant CF alleles were given a homopolymer T tail with terminal deoxyribonucleotidyltransferase and then immobilized on a nylon membrane. T-tail homopolymers were preferentially bound to the nylon, leaving the specific ASO sequences free to hybridize with amplified and radiolabeled exons 10 and 11 of a patient. These exons were simultaneously coamplified by a multiplex PCR and radiolabeled by random priming. ASO reverse dot blot currently appears to be the most efficient, rapid, and economic means of screening the population for CF mutations. This screening can detect nearly 80% of carriers and 64% of couples at risk and could prevent the birth of CF-affected infants in these families.

Thirteen mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been screened in a French sample of 185 cystic fibrosis (CF) patients, together with their respective associated RFLP haplotypes at the linked D7S23 locus (XV2C and KM19 markers). The respective frequencies of the mutations showed that 9 of them account for 80% of the CF chromosomes. Implications for prenatal diagnosis and heterozygote detection are defined and discussed. The well-known great excess of RFLP B marker within CF chromosomes is partially explained by two already characterized mutations highly associated with haplotype B: delta F508 and G542X. Similarly, the excess of haplotype D within CF chromosomes is partially explained by the association between delta I507 and this haplotype. These results may suggest the existence of two still untested or uncharacterized mutations, whose frequencies could be near 1%, one which would be associated with haplotype B and a second which would be associated with haplotype D. The possible cause of the specific association between most of the main different CF mutations and the RFLP haplotype B is discussed.

In the central nervous system, myelin proteolipid protein isoforms (PLP and DM20) play an essential structural role in myelination. It has been shown in several species that myelination is impaired by molecular defects resulting from single base mutations in the PLP gene. We have used DNA amplification by polymerase chain reaction to study the PLP gene coding regions from 17 patients in 15 unrelated families with similar Pelizaeus-Merzbacher phenotype. In one case amplification of peripheral nerve PLP/DM20 cDNAs revealed that a silent T----C transition was unrelated to the disease. In one family a nonsilent mutation was identified that leads to a phenylalanine substitution for valine-218 in PLP/DM20 proteins. We investigated the inheritance of the mutant allele in 19 subjects of this four-generation family and found a strict cosegregation of the Phe218 substitution with transmission and expression of the disease. The effect of the Val218----Phe mutation is discussed in the frame of a recently suggested topological model of PLP/DM20, according to which Val218 is part of an extracellular loop that connects the last two of four membrane-spanning alpha-helices.

The fragile X syndrome, a common cause of inherited mental retardation, is characterized by an unusual mode of inheritance. Phenotypic expression has been linked to abnormal cytosine methylation of a single CpG island, at or very near the fragile site. Probes adjacent to this island detected very localized DNA rearrangements that constituted the fragile X mutations, and whose target was a 550-base pair GC-rich fragment. Normal transmitting males had a 150- to 400-base pair insertion that was inherited by their daughters either unchanged, or with small differences in size. Fragile X-positive individuals in the next generation had much larger fragments that differed among siblings and showed a generally heterogeneous pattern indicating somatic mutation. The mutated allele appeared unmethylated in normal transmitting males, methylated only on the inactive X chromosome in their daughters, and totally methylated in most fragile X males. However, some males had a mosaic pattern. Expression of the fragile X syndrome thus appears to result from a two-step mutation as well as a highly localized methylation. Carriers of the fragile X mutation can easily be detected regardless of sex or phenotypic expression, and rare apparent false negatives may result from genetic heterogeneity or misdiagnosis.

To characterize mutations in the CYP21B gene that are responsible for congenital adrenal hyperplasia (CAH), DNA samples from 91 French patients have been studied by allelic-specific oligonucleotide hybridization and Southern blot analysis. Seven sites mostly found in the CYP21A pseudogene and deletions of the functional CYP21B gene have been screened. Gene conversions involving small DNA segments accounted for 57% of the tested mutations and probably cause 74% of the mutations responsible for the disease. Complete deletion of the CYP21B gene accounted for 18% of the CAH mutations in the whole sample and for 21% in the classical form of the disease. Three mutations were found associated with specific clinical forms of the disease: a G-C substitution in the seventh exon was associated with the late-onset form of the disease, and both an 8-bp depletion in the third exon and complete deletion of CYP21B were associated with the salt-wasting form.

Spanish families (n = 75) with at least one affected cystic fibrosis (CF) child were typed for restriction fragment length polymorphisms (RFLPs) by the probes XV2c, KM19, and pMP6d-9. These families were also studied at the 508 mutation site by the polymerase chain reaction method. We have studied the linkage disequilibrium between these markers and the CF mutations, the probable number of independent secondary CFX (non-delta F508) mutations, and the genetic differences between Spain and Western Europe.

French families (n = 129) with at least one cystic fibrosis (CF) affected child and 44 unrelated subjects from the general population were tested for the presence of the delta F508 mutation by the polymerase chain reaction. The delta F508/CF mutation ratio (CF: uncharacterised CF mutations) was tested in the CF families with and without meconium ileus. The association between delta F508 and CF mutations and restriction fragment length polymorphism haplotypes (XV2c and KM19) has been estimated; these data suggest that the CF chromosomes include a panel of independent and probably different mutations.

The prenatal diagnosis of cystic fibrosis is now routinely performed by using two probes tightly linked to the CF locus (XV2C and KM19). These probes have been shown to exhibit a strong linkage disequilibrium with the CF locus. Our data (103 families) have been pooled with other French data (237 families). They are consistent with the hypothesis of a unique ancestral mutation initially associated with a B (D1E2) restriction fragment length polymorphism (RFLP) haplotype, subsequently reassociated by cross-over with A, C or D haplotypes. Assuming such an hypothesis, the mutation is supposed to be 3000-6000 years old, depending on generation length and the true recombination ratio between the KM19 and CF loci. Up-to-date Spanish, Danish and Greek data are reported together with other previously published population data in order to discuss the geographic origin and age of the mutation in Europe. The action of selection in terms of heterozygote advantage and distorsion of segregation is discussed.

Correlations have been established between phenotype and karyotype in spontaneously aborted specimens. The criteria used to determine the phenotype were based on estimated developmental age and macroscopic examination of embryo and placenta, taking into account the morphological modifications due to in utero retention. The probability that a chromosomal aberration was the cause of the abortion is high when the developmental arrest occurred before 5-6 weeks and the expulsion was delayed. The frequency could be estimated at 75% when the developmental arrest was at 2-3 weeks, and 65% when the developmental stage reached 5-6 weeks. Main anatomical features are described permitting an attempt to assign the type of chromosomal anomaly by ultrasonographic examination.

Amniotic fluid porphyrins, biosynthesis of porphyrins by amniotic cells, and uroporphyrinogen III cosynthetase were studied after the 17th week of a pregnancy at risk for congenital erythropoietic porphyria (CEP). Only coproporphyrin was found in amniotic fluid. A diagnosis of CEP was ruled out by the demonstration of normal cosynthetase activity; biosynthesis of porphyrins was identical, not only in the porpositus and in control amniotic cells, but also in patients with CEP and in control skin fibroblasts.

By in situ hybridization of normal human chromosomes with a cloned genomic probe specific for the constant region of the lambda immunoglobulin genes, band 22q11 was preferentially labelled. In two cell lines with t(8;22) derived from Burkitt's lymphoma a strong signal was noted on the 8q+ chromosome derivative, indicating that the constant region of the lambda Ig gene cluster was translocated from chromosome 22 to chromosome 8. In addition, the signal observed on the 22q- derivative chromosome was stronger than the background in one of the two cell lines tested, but not in the other. The implications are that the break point in chromosome 22 in some cases lies within the Ig gene itself or between clusters of such genes, and that different cases have different break points.

We used a cloned cDNA probe for the B subunit of human aldolase (ALDB) and Southern blotting techniques to analyse DNA from a series of rodent X human somatic cell hybrids for the presence of specific ALDB-related sequences. Our results provide evidence for the assignment of the gene for ALDB to chromosome 9. Moreover, by direct gene dosage determination in two patients with chromosome 9 unbalanced rearrangements and by in situ hybridization we refined the regional chromosomal assignment to 9q13----q32 and most probably to 9q21.3----9q22.2.

During a cytogenetic study of human spontaneous abortions, attempts were made to initiate cell lines from tissues of embryos with chromosomal anomalies. The rate of success of these attempts and the life-span of the cell cultures is correlated with the development attained by these aneusomic embryos.

HLA markers (A, B, C, DR loci) were determined for the members of 52 unrelated families with at least one child suffering from congenital adrenal hyperplasia due to 21 hydroxylase deficiency, permitting genotyping. The gene frequencies of the 52 index cases were compared with those obtained from the patients' normal haplotypes and with those of a control reference panel. No significant differences were observed, except a clear decrease in the frequency of HLA-B8 among the haplotypes that carry the gene for congenital adrenal hyperplasia.

Gene dosage studies yielded results consistent with the assignment of the locus for pyruvate kinase (PK3) to chromosome 15. The activity of seven cytoplasmic enzymes has been determined in fibroblast extracts from six trisomy 15 lines and 16 normal control lines. The fibroblast extracts from the trisomic patients had pyruvate kinase activity 57% higher than fibroblast extracts from control lines, while other enzyme activities were within the normal range of activity.

A first trimester prenatal diagnosis of adrenoleukodystrophy has been done on chorionic villi biopsy in the pregnancy of a carrier woman. Two different approaches allowed one to determine that the male fetus was affected: the linkage analysis of DNA from chorionic villi using the highly polymorphic probe St 14 and the determination of very long chain fatty acid levels in cultured chorionic villi.

The fragile-X mental retardation syndrome, one of the most prevalent chromosome X-linked diseases (approximately equal to 1 of 2000 newborn males), is characterized by the presence in affected males and in a portion of carrier females of a fragile site at chromosomes band Xq27. We have performed a linkage analysis in 16 families between the locus for the fragile-X syndrome, FRAXQ27, and two polymorphic DNA markers that correspond to the anonymous probe St14 and to the coagulation factor IX gene F9. Our results indicate that the order of loci is centromere-F9-FRAXQ27-St14-Xqter. The estimate of the recombination fraction for the linkage F9-FRAXQ27 is 0.12 (90% confidence limits: 0.044-0.225) and 0.10 for FRAXQ27-St14 (90% confidence limits: 0.040-0.185). Recombination between St14 and F9 does not appear to be significantly different in normal and fragile-X families. The two flanking probes were used for diagnosis of the carrier state and for detection of transmission of the disease through phenotypically normal males. They should also allow first-trimester diagnosis with a reliability of about 98% in 40% of the families. Used in conjunction with the cytogenetic analysis, the segregation studies with both probes should improve the genetic counseling for the fragile-X syndrome and should be useful for the formal genetic analysis of this unique disease.

Prenatal diagnosis of cystic fibrosis was performed in 200 pregnancies with a 1-in-4 risk, and was based on significant modifications in amniotic fluid taken at 17, 18, 19 weeks of pregnancy, of six enzymatic assays: gamma-glutamyl-transpeptidase, aminopeptidase M, and alkaline phosphatase (total and isoenzymes). On the basis of normal values, normal outcome was predicted in 135 pregnancies reaching term, all the babies were normal. On the basis of significantly abnormal enzymatic values, an affected fetus was predicted in 56 pregnancies, 53 were terminated, and 3 went to term; the infants were affected. There were discrepancies in enzymatic values in nine cases, in eight cases normal outcome was predicted, six babies were normal and two were affected; in one case an affected baby was predicted, the pregnancy went to term and the baby is normal. Criteria giving evidence for cystic fibrosis in fetuses have been described: macroscopic observation of a typical meconium ileus, significant increase of albumin content in the meconium, and PAS-positive mucus-like material in some pancreatic acini. Using these criteria, diagnosis of cystic fibrosis has been confirmed in all the examined fetuses. The recurrence rate of cystic fibrosis was 22.5% in 147 diagnoses in which the index case had cystic fibrosis without a history of meconium ileus at birth, but was 47.5% when the index case had meconium ileus. The results of the study suggest that prenatal diagnosis of cystic fibrosis can be performed with an accuracy of 98%.

Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.

A 32-year-old woman, who presented with four spontaneous abortions, was found to have a balanced translocation: 46,XX,t(13:18)(q34:q11). In the last pregnancy an amniocentesis was done. Abnormal constitution of the fetus had been detected: 47,XY,t(13:18)(q34:q11) + 18, and an abortion was induced. Examination of the fetal tissue confirmed the finding. The fetus showed the characteristics of Edward's syndrome. Through the patient's pedigree it was discovered that balanced translocation appeared in three generations.