The trophoblastic epithelial barrier is not infected in full-term placentae of human immunodeficiency virus-seropositive mothers undergoing antiretroviral therapy.

To study the mechanism of the placental barrier function, we examined 10 matched samples of term placentae, cord blood, and maternal blood obtained at delivery from human immunodeficiency virus (HIV)-infected mothers with children diagnosed as HIV negative in Sweden. All placentae were histologically normal, and immunochemistry for HIV type 1 p24 and gp120 antigens was negative. Highly purified trophoblasts (93 to 99% purity) were negative for HIV DNA and RNA, indicating that the trophoblasts were uninfected. Although HIV DNA was detected in placenta-derived T lymphocytes and monocytes, microsatellite analysis showed that these cells were a mixture of maternal and fetal cells. Our study indicates that the placental barrier, i.e., the trophoblastic layer, is not HIV infected and, consequently, HIV infection of the fetus is likely to occur through other routes, such as breaks in the placental barrier.

A second locus for autosomal dominant myopathy with proximal muscle weakness and early respiratory muscle involvement: a likely chromosomal locus on 2q21.

We recently mapped a locus for a new variant of autosomal dominant myopathy (Swedish families) with proximal muscle weakness, early respiratory muscle involvement, and unique muscle biopsy findings to chromosomal region 2q24-31. In this study, a French family with a similar clinical phenotype and pathology (muscle biopsy) was investigated to see whether the disease gene associated with the myopathy is mapped to the same region as the one in the Swedish families; however, chromosomal region 2q24-q31 was completely excluded. In order to localise the disease gene for the French family, a genome-wide scan was performed using polymorphic microsatellite markers. A maximum two-point lod score of 2.11 (the highest lod score that can be achieved in this family) was obtained for the markers in the region between D2S1272 and D2S1260, spanning 4 cM. This result suggests that the gene responsible for the French form is likely to be located on chromosome 2q21.

Simultaneous analysis of expression of the three myotonic dystrophy locus genes in adult skeletal muscle samples: the CTG expansion correlates inversely with DMPK and 59 expression levels, but not DMAHP levels.

The causative mutation in the majority of cases of myotonic dystrophy has been shown to be the expansion of a CTG trinucleotide repeat, but the mechanism(s) by which this repeat leads to the very complex symptomatology in this disorder remains controversial. We have developed a highly sensitive and quantifiable assay, based on competitive RT-PCR, to test the hypothesis that the expansion disrupts the expression of the genes in its immediate vicinity, DMPK, 59 and DMAHP. In order to avoid cell culture-induced artifacts we performed these experiments using adult skeletal muscle biopsy samples and analysed total cytoplasmic poly(A)+mRNA levels for each gene simultaneously, as this is more physiologically relevant than allele-specific levels. There was considerable overlap between the expression levels of the three genes in myotonic dystrophy patient samples and samples from control individuals. However, in the myotonic dystrophy samples we detected a strong inverse correlation between the repeat size and the levels of expression of DMPK and 59. This is the first report of a possible effect of the CTG expansion on gene 59. Our results indicate that whilst a simple dosage model of gene expression in the presence of the mutation is unlikely to be sufficient in itself to explain the complex molecular pathology in this disease, the repeat expansion may be a significant modifier of the expression of these two genes.

CTG-repeat length in distal and proximal leg muscles of symptomatic and non-symptomatic patients with myotonic dystrophy: relation to muscle strength and degree of histopathological abnormalities.

Myotonic dystrophy (DM) is an autosomal dominant, multisystemic disorder with a variable phenotypic expression including muscle weakness and myotonia. The muscle wasting is most marked in distal limbs and in facial and neck muscles, although proximal limb muscles become affected as the disease progresses. The CTG-trinucleotide-repeat expansion associated with myotonic dystrophy is usually larger in muscle tissue than in leukocytes. It is unclear whether the repeat length itself bears any relation to the differences in the degree of weakness and atrophy between different muscles. We therefore analysed CTG-repeat lengths in blood and in proximal (m. vastus lateralis) and distal (m. tibialis anterior) muscles of patients with DM (n = 4) and non-symptomatic carriers of the mutant DM allele (n = 2) using conventional Southern blot hybridization. Muscle strength and histopathological abnormalities were evaluated for each muscle. In patients with clinical symptoms, the degree of paresis and morphological abnormalities was markedly more pronounced in m. tibialis anterior than in m. vastus lateralis. In these individuals, the CTG-repeat length was larger in muscles than in leukocytes, whereas in the two non-symptomatic carriers no difference could be detected. Furthermore, there was no clear difference in the repeat length between the two muscles in any of the patients. In conclusion, the selective muscular weakness and atrophy in DM do not seem to be related to differences in CTG-repeat length between different muscles.

Screening for mutations in candidate genes for hypospadias.

Hypospadias. a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5alpha-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerase chain reaction (PCR) amplification of the AR, WTI and 5alpha-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5alpha-reductase gene. In summary, mutations in the WT1, AR and 5alpha-reductase genes are not common causes of isolated hypospadias.

Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B).

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease.

Low frequency of RET mutations in Hirschsprung disease in Sweden.

Hirschsprung disease is a congenital malformation, where absence of intramural ganglia in the hindgut results in a defect in the coordination of peristaltic movement. This leads to ileus in the newborn or, more often, constipation in children and adults. The disease affects one in 5000 live births. Siblings of affected cases are at an increased risk (4%) of developing the disease. Among cases. males are affected more often than females. The first major susceptibility gene for Hirschsprung disease is the RET proto-oncogene on 10q11.2. Germline RET mutations in Hirschsprung disease are mainly inactivating, and have been reported to account for up to 20 and 50% of sporadic and familial cases, respectively. We have screened Swedish population-based samples from 62 sporadic cases and seven familial cases of Hirschsprung disease with single strand conformation polymorphism (SSCP), and found five mutations.

Welander distal myopathy--an overview.

Welander distal myopathy has an autosomal dominant inheritance and a late onset. The onset of symptoms is in the hands and gradually distal muscles of the lower extremities are involved. The most-affected muscles are the long extensors of the hands and feet. CK-values are normal or slightly elevated. There is never any cardiac involvement in Welander distal myopathy. Neurophysiological findings are of both myopathic and neuropathic character. Histopathological findings in muscle biopsies are mainly of myopathic type and include rimmed vacuoles which correspond to autophagic vacuoles on the ultrastructural level. Tubulo-filamentous inclusions with a diameter of 16-21 nm, i.e. of the same type as found in patients with Inclusion Body Myositis, are found in the sarcoplasm and in myofibre nuclei. A neurogenic component in Welander distal myopathy has been suggested, on the grounds of a sensory dysfunction, neuropathic findings on neurophysiology and muscle biopsy and a decrease of A-delta nerve fibres on sural nerve biopsy. Genetic analysis has excluded linkage to other defined distal myopathies and hereditary Inclusion Body Myopathy loci.

A novel missense mutation Ile538Val in the fibroblast growth factor receptor 3 in hypochondroplasia. Mutations in brief no. 122. Online.

Hypochondroplasia and achondroplasia are skeletal dysplasias, characterized by autosomal dominant inheritance and disproportionate short stature, which occurs mainly due to growth failure of the extremities. Both dysplasias have been mapped to fibroblast growth factor receptor 3 (FGFR3) gene. For hypochondroplasia, two point mutations, both responsible for the Asn540Lys substitution in the region coding the tyrosine kinase domain have been reported. Here we report an A to G transition at position 1651, predicting an Ile538Val substitution in the FGFR3, in hypochondroplasia. The substitution is found in a swedish family with three affected members. The criteria for hypochondroplasia were disproportionate short stature and radiological evidence of shortened long bones and decrease or absence of normal increase in interpedicular distances of the lumbar column. The mutation was detected by direct sequencing and restriction enzyme Tai I digestion. The base change was not found in the FGFR3 genes of unaffected members of the family nor in seventy-five unrelated unaffected individuals, suggesting that it was not a polymorphism. The Ile538Val substitution is a conservative amino acid change (a hydrophobic amino acid incorporated for another hydrophobic amino acid). Nevertheless, it is located in the stretch of nine amino acids, which is highly conserved among all the human fibroblast growth factor receptors. Considering the location of this substitution and the segregation with the phenotype in this family, we propose that it is a causative mutation of hypochondroplasia. It is difficult to establish whether the Ile538Val substitution is rare in hypochondroplasia patients or whether the individuals, who have a moderate degree of short stature, rarely seek medical help for the short stature and consequently are rarely diagnosed as affected by hypochondroplasia.

C4342T-mutation in the SCN4A gene on chromosome 17q in a Swedish family with paramyotonia congenita (Eulenburg)--correlations with clinical, neurophysiological and muscle biopsy data.

Genetic analysis of the adult muscle sodium channel alpha-subunit, SCN4A gene on chromosome 17q, was performed by means of PCR technique in a Swedish family with paramyotonia congenita (Eulenburg) (PMC). The mutation was found in four family members and consisted of a C to T transition affecting the fourth domain of the sodium channel protein. This mutation has earlier been described in other families with paramyotonia congenita. All family members carrying the mutation had cold-induced paradoxical myotonia, myotonic bursts on EMG, and a type IIB atrophy on muscle biopsy. Three of them had slight CK elevation and two had episodes of paralysis. On the basis of clinical findings in this family, persistent proximal muscle weakness, myopathic EMG abnormalities, a type IIB atrophy on muscle biopsy and no symptoms but other signs of muscle affection, were earlier suggested as clinical features of PMC. However, genetic analysis revealed that family members with these symptoms and findings did not have the mutation, indicating that these features are not due to PMC.

Variation of CTG-repeat number of the DMPK gene in muscle tissue.

Myotonic dystrophy (DM) is associated with an unstable expansion of CTG repeats located in the 3' untranslated region of a protein kinase-encoding gene (DMPK) on chromosome 19 (19q13.3). The CTG repeat number varies between 5 and 37 in lymphocytes of normal individuals, whereas DM patients may have expansions from 50 to several thousand copies. Although the CTG expansions related to myotonic dystrophy (DM) are usually larger in muscle compared to peripheral blood, the variation in repeat number in non-dystrophic muscle is not known. In order to investigate if there is a variation, the CTG-repeat number was determined in percutaneous muscle biopsies obtained from 86 individuals without any muscle disorder or with a neuromuscular disorder without any clinical or histopathological signs of DM. The number of CTG repeats varied between 5 and 28, this being within the normal range reported for peripheral blood. A major sharp peak at n = 5 (27%) and a broader peak at n = 8-17 (56%) with peak values at n = 12 and 14 (11 and 14%, respectively) were observed. Alleles with 19 or more repeats amounted to 17% with a small peak at n = 20 and 21 (6 and 4%, respectively). It is concluded that the normal variation of CTG-repeat number in skeletal muscle is within the range found in peripheral blood, although there is a slight shift in the overall frequency distribution towards alleles with CTG repeat numbers in the higher range.

Treatment of adrenoleukodystrophy with bone marrow transplantation.

Three children with adrenoleukodystrophy (ALD) underwent allogeneic bone marrow transplantation (BMT) between 1992 and 1993. The first boy had attention deficits, marked neuropsychological deficits and widespread demyelination in the frontal lobes on MRI before transplantation. Four years later he has mentally deteriorated and the demyelination on MRI has progressed. The second boy had no symptoms but had white matter lesions on MRI when diagnosed. He was regularly followed with MRI and neuropsychological investigations until BMT 18 months later. A progress of the lesions was noted on the initial MRI investigations, and 4 months before BMT a worsening of deficits in attention and kinaesthetic praxis could be observed. He rapidly deteriorated after the transplantation and died 18 months later. Both PCR and in situ hybridization confirmed the presence of donor cells in the brain. The third boy had no symptoms but white matter lesions on MRI when diagnosed. The neuropsychological tests remained normal but a slight progress was observed on MRI just before transplantation. This boy is still healthy 3.5 years after BMT. BMT as treatment for ALD has to be considered very early, even if a child without symptoms but signs of demyelination on MRI, if a suitable donor is available.

Characterization and regulation of the nonerythroid porphobilinogen deaminase promoter.

The non-erythroid porphobilinogen deaminase (E.C. 4.3.1.8) promoter was investigated according to sequence changes and transcriptional activity. The minimal promoter sequence required for maximal transcription, was localized by deletion mapping to -243 to -115 relative to the translational start site. A negative transcriptional element was found between -55 and +1, indicating a repression mechanism. A new polymorphism was identified, at position -235 within the minimal promoter, both in acute intermittent porphyria patients and healthy control subjects. The polymorphic variants did not affect expression of cloned promoter fragments under the conditions used.

Achondroplasia in Sweden caused by the G1138A mutation in FGFR3.

Achondroplasia, an autosomal dominant inherited disorder, is one of the most common forms of skeletal dysplasia resulting in disproportionate extreme shortness. Recently, two point mutations, both affecting nucleotide 1138 in the fibroblast growth factor receptor type 3 (FGFR3) gene, were found to be the cause of the disorder. We investigated DNA from 16 Swedish patients with achondroplasia for the presence of these mutations. All patients were found to be heterozygous for the G to A transition at nucleotide 1138. Our data thus support previous reports showing a striking genetic homogeneity, in that almost all achondroplasia patients have the FGFR3 G380R mutation at the protein level.

No evidence of WT1 gene mutations in children with congenital diaphragmatic hernia.

Children with congenital diaphragmatic hernia are born with a defect of the diaphragm that usually leads to a herniation of abdominal organs up into the thoracic cavity on the same side. The condition is often fatal, usually because of concomitant lung hypoplasia. The cause of this condition is unknown. The majority of cases are sporadic, but familial aggregation has been observed, indicating a genetic background. The tumor suppressor gene WT1 is involved in normal early urogenital development, and it is expressed in the mesothelium in the early human fetus. Recently, knockout mice homozygous for WT1 gene mutations were constructed. The embryos died between days 13 and 15 of gestation. Besides serious urogenital and thoracic malformations, the mice had defects in the diaphragm that caused herniation of lung tissue into the abdomen. These findings prompted the authors to screen for WT1 gene mutations in 27 children who had congenital diaphragmatic hernia. Using exon-per-exon polymerase chain reaction (PCR) amplifications and denaturing gradient gel electrophoresis, no WT1 mutations were detected. Southern blot analysis did not show any large rearrangements in the WT1 gene. These results exclude WT1 gene mutations as a major etiological factor for the isolated diaphragmatic defect. However, it is possible that other genes in the WT1 pathway are involved in this defect.

Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies.

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

Constitutional and somatic mutations in the WT1 gene in Wilms' tumor patients.

Wilms' tumor development, like most tumors, involves multiple genetic alterations affecting diverse genes. Only one of these has thus far been identified, the Wilms' tumor 1 (WT1) gene on 11p13, which functions as a tumor suppressor gene. We assessed the involvement of the WT1 gene constitutionally and somatically in 26 Wilms' tumor patients. Of these, the clinical data suggest a constitutional pre-disposition in 12 bilateral cases and 7 cases with early onset. We employed Southern blot analysis and PCR-based markers for analyses of somatic allelic losses in chromosome bands 11p13, 11p15 and 16q and screened for point mutations in exons 2-10 of the WT1 gene with denaturing gradient gel electrophoresis (DGGE). Of the 12 cases with multiple tumors, 1 exhibited a constitutional 11p13 deletion and a somatic stop mutation in exon 4 of the WT1 gene and 2 harbored constitutional mutations in the WT1 gene: a pre-mature stop codon in exon 6 in a boy with bilateral cryptorchidism and bilateral Wilms' tumors and an intragenic deletion in a girl with bilateral WT. Three additional bilateral tumors displayed WT1 rearrangements or allelic losses with 11p13 markers. Four of 7 patients with an early onset of unilateral tumor had losses of 11p13, though no WT1 mutations were detected. Two of the remaining cases that did not show any somatic or constitutional 11p13 alterations had Beckwith-Wiedemann syndrome, known to involve the 11p15 region.

Absence of mutations in the WT1 gene in patients with XY gonadal dysgenesis.

The WT1 gene is normally expressed during gonadal development and specific mutations in heterozygous form cause Drash syndrome, characterized by male pseudohermaphroditism and gonadal dysgenesis, renal failure and a predisposition for Wilms' tumour. These observations prompted us to test whether WT1 mutations are involved in isolated gonadal dysgenesis, being the most severe form of disturbance in gonadal differentiation. We studied 27 cases of 46,XY females with gonadal dysgenesis who had previously been screened for and found not to carry SRY gene mutations. We performed mutational screening of the WT1 gene with denaturing gradient gel electrophoresis. In one of these patients, a heterozygous point mutation in exon 8 was found. This mutation has previously been described in Drash syndrome and re-evaluation of the clinical data confirmed this diagnosis. Based on these results, we conclude that isolated gonadal dysgenesis is not caused by mutations in the WT1 gene.