B. Rafael Elejalde

Up-Regulation of WNT-4 Signaling and Dosage-Sensitive Sex Reversal in Humans

Wnt-4, a member of the Wnt family of locally acting secreted growth factors, is the first signaling molecule shown to influence the sex-determination cascade. In mice, a targeted deletion of Wnt-4 causes the masculinization of XX pups. Therefore, WNT-4, the human homologue of murine Wnt-4, is a strong candidate gene for sex-reversal phenotypes in humans. In this article, we show that, in testicular Sertoli and Leydig cells, Wnt-4 up-regulates Dax1, a gene known to antagonize the testis-determining factor, Sry. Furthermore, we elucidate a possible mechanism for human XY sex reversal associated with a 1p31-p35 duplication including WNT-4. Overexpression of WNT-4 leads to up-regulation of DAX1, which results in an XY female phenotype. Thus, WNT-4, a novel sex-determining gene, and DAX1 play a concerted role in both the control of female development and the prevention of testes formation. These observations suggest that mammalian sex determination is sensitive to dosage, at multiple steps in its pathway.

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

A multicenter investigation with D-FISH BCR/ABL1 probes

Twenty-eight laboratories evaluated a new fluorescence in situ hybridization (FISH) strategy for chronic myeloid leukemia. In a three-part study, bcr/abl1 D-FISH probes were used to study bone marrow specimens. First, laboratories familiarized themselves with the strategy by applying it to known normal and abnormal specimens. Then, collectively the laboratories studied 20 normal and 20 abnormal specimens blindly and measured workload. Finally, each laboratory and two experts studied six serial dilutions with 98-0% abnormal nuclei. Using the reported normal cutoff of < 1% abnormal nuclei, participants reported no false-negative cases and 15 false-positive cases (1-6.6% abnormal nuclei). Results provided by participants for serial dilutions approximated the expected percentages of abnormal nuclei, but those from the experts exhibited greater precision. The clinical sensitivity, precision, nomenclature, workload, recommendations for training, and quality assurance in methods using D-FISH in clinical practice are discussed.

Hallervorden-Spatz disease

We had the opportunity to study a family, five of whose members were affected by the Hallervorden‐Spatz disease (three males and twin girls). The characteristics of the condition were analyzed and compared with those cases considered by other authors to be affected by the condition. Intrafamilial and interfamilial variations were analysed, and it was the latter that contributed most to the overall variation of the condition. It was clearly established from the reported cases and our family that this is an autosomal recessive condition (P> 0.23 ± 0.08). It is suggested that the condition probably originated in Europe and that it is caused by an inborn error of metabolism related to neuromelanin and the dopaminergic system.

Linear facial skin defects associated with microphthalmia and other malformations, with chromosome deletion Xp22.1

nous porphyrins after exogenous administration of ALA. The same studies also showed that inactivation of ALA-treated T cell lymphoma Eb-Esb cells occurred after photoexcitation by visible light. Malik et a1.9 speculated that the accumulation of porphyrins in lymphoma cells may be caused by a lack of ferrochelatase, which is needed for the insertion of iron into the porphyrin ring in the mitochondria of leukemic cells. Thus the efficiency of PDT after ALA photosensitization in MF may be based on direct cytotoxic mitochondrial damage to T lymphocytes in the skin. Our results showing the efficacy of ALA photosensitization in PDT for MF suggest further studies to determine the optimal ALA and light dose combination, frequency of treatment, and duration of remission. Furthermore, the findings also suggest that, provided an appropriate light source becomes available for exposing larger body areas to visible light, topical3, 6, 7 or systemic8, 12 ALA photosensitization may be effective in PDT for more widespread and extensive MF.

Determination of cholinesterase and acetylcholinesterase in amniotic fluid. Uses in prenatal diagnosis and quality control.

The determination of acetylcholinesterase (AChE) has been shown to be as specific as alphafeto‐protein (AFP) for the prenatal detection of open neural tube defects although AFP remains the method of choice. This paper describes a semi‐automated technique for the analysis of acetylcholinesterase in amniotic fluid that: A) reduces the cost of the procedure; B) allows for a larger number of samples to be run at a time; and C) provides for more accurate and reproducible procedures and results. Six fetuses with neural tube defects (2 with gastroschisis and 3 where one twin was dead) were detected and found to have elevated AChE, TChE and 2 bands by electrophoresis. Quality control procedures using both pure enzyme and amniotic fluid with low and high levels of the enzyme are described. The analysis of 340 amniotic fluids of normal pregnancies indicates that the normal value for AChE is 5.17 ± 2.63 mU/ml (97% confidence interval for the mean 4.84‐5.49 mU/ml. A group of 27 abnormal pregnancies provides evidence that fetal vomiting and regurgitation, fetal demise, multiple cysts syndrome, idiopathic IUGR, arthrogryposis multiplex, hydrocephaly (stenosis of aqueductus), trisomy 21, trisomy 18, hydronephrosis, pyloric stenosis, heart malformation, ectopia cordis and multiple gestation produce elevated levels of pseudocholinesterase (PChE) in amniotic fluid. The use of pseudocholinesterase levels in amniotic fluid for prenatal diagnosis is proposed and discussed in view of its elevated levels in abnormal pregnancies where AChE is normal. The normal values for PChE are 23.86 mU/ml (mean) and 5.83 for standard deviation. Electrophoretic analysis was performed on all samples with values higher than one standard deviation above the mean. The AChE band was found only in fetuses with open neural tube defects and pyloric stenosis.

Neuroectodermal Melanolysosomal Disease

Publisher Summary This chapter describes neuroectodermal melanolysosomal disease (NEMLD). NEMLD is characterized by abnormal hair color, severe dysfunction of the central nervous system—profound mental, developmental, and behavioral retardation—abnormal intracytoplasmic inclusions in all tissues studied, and abnormally formed melanosomes. The condition appears to be caused by the pleiotropic effects of an autosomal recessive gene in its homozygous state, producing various phenotypic effects in different tissues. The unusual hair color probably results from the melanocytes in the hair bulb producing melanin in the presence of the different substrates. The color of the hair is probably produced by two factors: (1) the distribution of the melanosomes in the hair shaft, and (2) the diffraction, refraction, and absorption of light by both the pigment granules and the hair matrix. Abnormalities of the dopaminergic pathway are known to be associated with an abnormally functioning central nervous system. In NEMLD, the hypomelanized melanosomes in the skin, the lysosomal inclusions in the fibroblasts, and the lymphocytes and bone marrow cells can be considered authophenes. No specific treatment is available for NEMLD. The infants and children require the specialized care appropriate for children with profound mental retardation and cerebral palsy.

Analysis of the human fetal skeleton and organs with xeroradiography

We have used xeroradiography to study normal and abnormal fetuses including some with anencephaly, hydrocephalus, spina bifida, osteogenesis imperfecta (type IV), Jeune syndrome, radial aplasia, thanatophoric dysplasia, and Pena-Shokeir syndrome. Xeroradiography images the lines of ossification and epiphyses in great detail, shows ossification, and reveals abnormalities that alter bone modeling as seen in Jeune syndrome, osteogenesis imperfecta (type IV), and thanatophoric dysplasia. This technique can be used successfully to examine soft tissues and organs. It can also be used in combination with contrast materials to identify the lateral ventricles, the cardiovascular, gastrointestinal, urinary, and respiratory systems, and the cavities (pleural and peritoneal) of the fetal body.

Fetal phenotypic analysis

Phenotypic analysis has been practised in many different ways, the most common being the recognition of normal and abnormal physical and biochemical characteristics and their patterns of inheritance. As different tools became available to analyze the characteristics of individuals the study of the phenotype and consequently of the genotype became more sophisticated. By the mid 1970’s ultrasonography became a major tool in the analysis of human disease, and more recently it has contributed to the analysis of the human fetal phenotype in utero. This method, when used in conjunction with cytogenetic and biochemical analysis of the amniotic fluid, constitutes the main thrust of genetic prenatal diagnosis today. This paper describes and proposes the systematic use of ultrasonography, in conjunction with biochemical and cytogenetic tests to examine pregnancies at risk of congenital and inherited diseases and those that show signs of abnormality. We have examined 1500 fetuses and have detected conditions that affect only one system, like the skeletal dysplasias (osteogenesis imperfecta, thanatophoric dysplasia, diastrophic dwarfism, achondrogenesis I, Jeune syndrome and many others), neural tube defects, gastroschisis, multiple congenital malformations syndromes (Vater, Vacterl, Weyers olygodactyly, Meckel syndrome, and others). The analysis of the functionality of the fetus is one of the major achievements of genetic prenatal diagnosis by ultrasonography.

Determination of the H-Y antigen in amniotic cells. Its use in prenatal diagnosis.

SummaryThis paper describes the determination of the H-Y antigen in cells obtained from amniotic fluids of two male and two female fetuses found to be normal at birth, and from another fetus that was suspected to have a karyotype 46,XY,t(15;Y) by Q banding. Since the area of the Y chromosome where the H-Y antigen gene appears to be located on the chromosomal material was suspected to be involved in the translocation, it was thought that this fetus could have a double dose of H-Y antigen as seen in 47,XYY individuals. The fetus was found to have a normal dose of the antigen and a normal 46,XY karyotype. The extra material on chromosome 15 was found to be NOR (nuclear organizer region) and to be present in his fathers karyotype. The possibility of this determination in other inherited conditions is discussed.The normal H-Y antigen was an important element in the counseling of this family and helped to rule out the suspected chromosome abnormality. The determination of H-Y antigen is shown to be reliable and simple in amniotic cells, both cultured and directly obtained from the fluid. Its determination appears to be useful in the prenatal determination of other conditions like campomelic dysplasia and Swyers syndrome.