Larry J. Shapiro

Human and mouse amelogenin gene loci are on the sex chromosomes

Enamel is the outermost covering of teeth and is the hardest tissue in the vertebrate body. The enamel matrix is composed of enamelin and amelogenin classes of protein. We have determined the chromosomal locations for the human and mouse amelogenin (AMEL) loci using Southern blot analyses of DNA from human, mouse, or somatic cell hybrids by hybridization to a characterized mouse amelogenin cDNA. We have determined that human AMEL sequences are located on the distal short arm of the X chromosome in the p22.1----p22.3 region and near the centromere on the Y chromosome, possibly at the proximal long arm (Yq11) region. These chromosomal assignments are consistent with the hypothesis that perturbation of the amelogenin gene is involved in X-linked types of amelogenesis imperfecta, as well as with the Y-chromosomal locations for genes that participate in regulating tooth size and shape. Unlike the locus in humans, the mouse AMEL locus appears to be assigned solely to the X chromosome. Finally, together with the data on other X and Y chromosome sequences, these data for AMEL mapping support the notion of a pericentric inversion occurring in the human Y chromosome during primate evolution.

Cloning and expression of steroid sulfatase cDNA and the frequent occurrence of deletions in STS deficiency: Implications for X-Y interchange

Human STS is a microsomal enzyme important in steroid metabolism. The gene encoding STS is pseudoautosomal in the mouse but not in humans, and escapes X inactivation in both species. We have prepared monoclonal and polyclonal antibodies to the protein which has been purified and from which partial amino acid sequence data have been obtained. cDNA clones containing the entire coding sequence were isolated, sequenced, and expressed in heterologous cells. Variable length transcripts have been shown to be present and due to usage of alternative poly(A) addition sites. The functional gene maps to Xp22.3-Xpter and there is a pseudogene on Yq suggesting a recent pericentric inversion. Absence of STS enzymatic activity occurs frequently in human populations and produces a visible phenotype of scaly skin or ichthyosis. Ten patients with inherited STS deficiency were studied and eight had complete gene deletions. The possibility that STS deficiency results from aberrant X-Y interchange is discussed.

METACHROMATIC LEUKODYSTROPHY WITHOUT ARYLSULFATASE A DEFICIENCY

Summary: Two siblings of consanguinous parents were noted to have a neurologic syndrome marked by developmental delay, regression of psychomotor performance, marked spasticity and progressive central nervous system degeneration. Markedly delayed nerve conduction times and a sural nerve biopsy which demonstrated changes typical of metachromatic leukodystrophy (MLD) were evident. Impairment of sulfated glycolipid metabolism was documented by analysis of glycospingolipid in urinary sediment. In spite of these findings, activities of arylsulfatase A and cerebroside sulfatidase in white blood cells and cultured skin fibroblasts were near normal. However, when intact growing fibroblasts were loaded with 35SO4-sulfatide a clear defect in sulfatide cleavage, comparable to that seen in MLD patients, was observed. Thus, these patients represent a new form of sulfatide storage disease – MLD characterized by intact enzyme activity in cell homogenates but defective sulfolipid metabolism in vivo and in intact fibroblasts.Speculation: Since cell homogenates from these patients can cleave sulfatide in the presence of detergents while the patients themselves and their intact cells cannot, soire explanation other than decreased activity of the relevant lysosomal enzyme must be invoked to explain this storage disease. The two most plausible hypotheses are that either these patients have a defect which prevents enzyme and substrate interaction in the proper subcellular location, or that these patients are missing the putative glycoprotein ″activating factor″ necessary for sulfolipid hydrolysis in vivo.

Steroid Sulfatase Deficiency

Summary: Placental steroid sulfatase deficiency is a genetic disorder only recently reported in the medical literature. Most documented cases of placental sulfatase deficiency have been marked by delay in onset of labor, lack of cervical dilatation, and relative refractoriness of oxytocic agents and amniotomy. We have studied the placenta, cultured fibroblasts, and amniotic fluid cells from an affected patient. The activities of estrone sulfatase, pregnenolone sulfatase, dehydrocpiandrosterone sulfatase, and arylsulfatase C in the placenta from the patient were severely deficient. Arylsulfatases A and B were present at levels within the normal range for this tissue.Fibroblast dehydroepiandrosterone sulfatase activity was virtually absent in the patients cells and present at normal levels in individuals with a variety of lysosomal disorders. It would thus appear that the mutation responsible for steroid sulfatase deficiency is genetically and biochemically distinct from those involved in the lysosomal sulfatase deficiency states. The cell culture studies further suggest that the defect is a generalized one which should be detectable in midtrimester of pregnancy and may have phenotypic consequences in later postnatal life.Speculation: Further clinical and in vitro studies of patients with this rare inborn error of metabolism should yield information regarding the role of steroid sulfatase in normal metabolism, and in the progress of normal parturition. Once the mechanism by which the level of activity of this enzyme exerts an effect upon labor and delivery is known, appropriate pharmacologic invervention might provide a means for the inhibition of premature labor.

Orally Administered Liposome-Entrapped Insulin in Diabetic Animals

We investigated the effects of liposome-entrapped insulin (LEI) administered orally one-half to one-tenth of previously reported doses, on plasma glucose and insulin in the spontaneously diabetic BB Wistar rat and in alloxan-induced diabetic rabbits. Incorporation of insulin within the liposome fraction ranged from 15 to 23%. Radioimmunoassay of Triton X-100 treated LEI yielded insulin values in high agreement (82 +/- 10%) with those predicted based on estimated incorporation. Whereas insulin alone, or liposomes devoid of insulin had no effect, LEI 5 U/kg significantly reduced glucose and raised insulin in 54% of rats (13 of 24) and 67% of the rabbits (6 of 9). Among the rats that responded, blood glucose fell from a basal of 318 +/- 21 mg/dl to a nadir of 186 +/- 22 mg/dl at 2 h (p less than 0.001); values at 1, 2 and 4 h were all significantly less (58-69%) than basal. Similarly, glucose declined significantly for 3 h post LEI in the rabbits while IRI rose from 30 +/- 7 micro U/ml to a peak of 399 +/- 75 micro U/ml at 1 h (p less than 0.001); values at 2 and 3 h remained significantly elevated. Some batches of LEI failed to reduce glucose despite apparently adequate incorporation, while even with effective batches some animals failed to respond. Thus, although orally administered LEI can be effective, their stability and effectiveness are not completely predictable.

Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in progressive muscular dystrophies.

PK and CPK have been determined in the serum from 208 individuals including 70 normal controls (61 adults and 9 children) and 138 patients with a variety of neuromuscular disorders. In adult controls the mean activity (+/- SE) for PK is 1.2 +/- 0.05 mumol/ml/h. In normal children PK activity was about twice as high as in normal adults and decreases with increasing age. In 26 patients with Duchenne dystrophy the range of serum PK was 4.0-150.4 and in 17 individuals with the Becker type, 3.0 to 148.7. All had elevated PK and CPK levels. Eighteen of 20 patients with the facio-scapulo-humeral (FSH) from of muscular dystrophy had increased PK while only 9 had elevated CPK. Regression analyses have shown an inverse correlation between PK levels and age (or degree of disability in DMD). Kinetic and electrophoretic studies indicate that the PK isozyme found in the serum from affected patients and from heterozygotes for the DMD gene is mainly the M1 type PK, which is the only PK isozyme found in skeletal muscle and brain and the major component from myocardium.

Steroid Sulfatase Deficiency and the Genetics of the Short Arm of the Human X Chromosome

It is considered axiomatic in human genetics that the study of relatively rare disorders may yield far more in dividends than might be anticipated based on the incidence of the condition in question. This has clearly been demonstrated in studies of human steroid sulfatase deficiency and the steroid sulfatase system during the past few years. Investigations of this infrequent human variation have led to expanded studies of sulfated steroid metabolism, the physiological control of epidermal keratinization, estrogen biosynthesis in pregnancy, testosterone biosynthesis, and the molecular mechanism of X-chromosome inactivation and the escape of inactivation of certain portions of the human X. In addition, the availability of a readily scoreable marker for the distal human short arm provides the potential basis for a number of observations regarding X/Y interchange involving this portion of the X and has raised a number of evolutionary issues as well. Further studies may help clarify several of these questions and substantially add to our understanding of a variety of human X-chromosome disorders, such as X aneuploid states, XX males, and true hermaphrodites.

Expression of epidermal growth factor in the rat kidney

SummaryThe renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.

Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in female relatives and patients with X-linked muscular dystrophies (Duchenne and Becker)

Determination of serum creatine phosphokinase (CPK) activity is often used in efforts to detect carriers of X-linked muscular dystrophies. We have recently demonstrated that another serum enzyme, pyruvate-kinase (PK) may also be of use in the diagnosis of patients affected with a variety of neuromuscular disorders. To evaluate the usefulness of this assay for carrier detection, a comparative study of serum PK and CPK activity was performed in 74 female relatives of patients affected with Duchenne (DMD) and Becker (BMD) muscular dystrophies. For obligate carriers of the DMD gene, 10 of 14 had elevated CPKs, 11 of 14 had elevated PKs and 12 of 14 had abnormal results for either of the two enzymes. Three of 16 mothers of isolated cases had increased serum CPK activity and 6 of 16 had increased PK activity (7 had elevation of at least one enzyme). These preliminary data suggest that the use of PK may enhance the capability to discriminate carriers for these X-linked recessive genes.

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.