Pharmacogenomics of insulin-like growth factor-I generation during GH treatment in children with GH deficiency or Turner syndrome.

Individual responses to growth hormone (GH) treatment are variable. Short-term generation of insulin-like growth factor-I (IGF-I) is recognized as a potential marker of sensitivity to GH treatment. This prospective, phase IV study used an integrated genomic analysis to identify markers associated with 1-month change in IGF-I (ΔIGF-I) following initiation of recombinant human (r-h)GH therapy in treatment-naïve children with GH deficiency (GHD) (n=166) or Turner syndrome (TS) (n=147). In both GHD and TS, polymorphisms in the cell-cycle regulator CDK4 were associated with 1-month ΔIGF-I (P<0.05). Baseline gene expression was also correlated with 1-month ΔIGF-I in both GHD and TS (r=0.3; P<0.01). In patients with low IGF-I responses, carriage of specific CDK4 alleles was associated with MAPK and glucocorticoid receptor signaling in GHD, and with p53 and Wnt signaling pathways in TS. Understanding the relationship between genomic markers and early changes in IGF-I may allow development of strategies to rapidly individualize r-hGH dose.

A pharmacogenomic approach to the treatment of children with GH deficiency or Turner syndrome.

OBJECTIVE: Individual sensitivity to recombinant human GH (r-hGH) is variable. Identification of genetic factors contributing to this variability has potential use for individualization of treatment. The objective of this study was to identify genetic markers and gene expression profiles associated with growth response on r-hGH therapy in treatment-naïve, prepubertal children with GH deficiency (GHD) or Turner syndrome (TS). DESIGN: A prospective, multicenter, international, open-label pharmacogenomic study. METHODS: The associations of genotypes in 103 growth- and metabolism-related genes and baseline gene expression profiles with growth response to r-hGH (cm/year) over the first year were evaluated. Genotype associations were assessed with growth response as a continuous variable and as a categorical variable divided into quartiles. RESULTS: Eleven genes in GHD and ten in TS, with two overlapping between conditions, were significantly associated with growth response either as a continuous variable (seven in GHD, two in TS) or as a categorical variable (four more in GHD, eight more in TS). For example, in GHD, GRB10 was associated with high response (≥ Q3; P=0.0012), while SOS2 was associated with low response (≤ Q1; P=0.006), while in TS, LHX4 was associated with high response (P=0.0003) and PTPN1 with low response (P=0.0037). Differences in expression were identified for one of the growth response-associated genes in GHD (AKT1) and for two in TS (KRAS and MYOD1). CONCLUSIONS: Carriage of specific growth-related genetic markers is associated with growth response in GHD and TS. These findings indicate that pharmacogenomics could have a role in individualized management of childhood growth disorders.

Mapping of a susceptibility gene for multiple sclerosis to the 51 kb interval between G511525 and D6S1666 using a new method of haplotype sharing analysis.

Multiple sclerosis (MS) is a complex disease that is partly genetic in origin. Although an association of MS with specific human leukocyte antigen (HLA) types has been known for almost 30 years, the nature of this relationship has remained unclear. Furthermore, genetic resolution sufficient to implicate a specific gene in the HLA region has not been achieved. Many loci in the HLA region have been found to be significantly associated with MS, which is largely explained by the extended haplotype sharing and varying marker informativity of the region. We have determined 248 haplotypes of MS patients from the population of the northern Netherlands and 226 haplotypes of their relatives as controls using a set of 22 microsatellite markers covering the HLA region. The data were analyzed using standard association methods and a new statistical method, haplotype sharing statistics (HSS). Haplotype sharing statistics determines the extent of haplotype sharing for all pairs of haplotypes of patients and of controls and calculates the difference in mean haplotype sharing between patients and controls. Haplotype sharing was found to be significantly greater among patients than among controls in a region of 1.1 Mb between markers G511525 and TNFalpha. The involvement of this region is also supported by association analysis and the transmission/disequilibrium test (TDT). Within this region, HSS, which is largely independent of association and TDT, indicated the interval of 51 kb between G511525 and D6S1666 as that most likely to contain a susceptibility gene for MS. As DQB1 is the sole gene known in this interval at present, the results of our analysis suggest that this gene plays a role in the pathogenesis of MS.

Up-regulation of estrogen receptor mRNA and estrogen receptor activity by estradiol in liver of rainbow trout and other teleostean fish.

Injection of estradiol (E2) into immature rainbow trout resulted in the induction of the hepatic vitellogenin gene mediated by the nuclear estrogen receptor (ER). Liver ER mRNA rose markedly on E2 treatment in three groups of trout kept at different temperatures. Only in the group kept at 4 degrees C did the total cellular ER, as measured by [3H]estradiol-binding activity in nuclear and cytosol fractions, parallel the ER mRNA level. In fish kept at 9 degrees C and 15 degrees C, the ratio of total ER activity to ER mRNA fell during chronic E2 treatment, probably reflecting translational of post-translational control mechanisms. Upregulation of ER mRNA also occurred in sea raven, sculpin, winter flounder, and Atlantic salmon after E2 treatment. Intrahepatic ER activity rose proportionately in Atlantic salmon kept at 6-9 degrees C but not in sea raven, sculpin, or flounder. We conclude that the regulation of ER expression in teleosts is complex and includes transcriptional, translational, and post-translational elements and is influenced by environmental temperature.

Alterations in expression, binding to ligand and DNA, and transcriptional activity of rearranged and wild-type retinoid receptors in retinoid-resistant acute promyelocytic leukemia cell lines.

All-trans retinoic acid (tRA), a naturally occurring ligand of the nuclear retinoic acid receptors (RARs), induces differentiation of leukemic cells and clinical complete remission in patients with acute promyelocytic leukemia (APL). This differentiation effect can also be seen in vitro in both fresh leukemic cells and in the unique permanent APL cell line, NB4. However, APL cells become resistant to RA-induced differentiation both in vitro and in patients. Although pharmacodynamic mechanisms of resistance have been reported, there is growing evidence that resistance both in patients, as well as in vitro, can be mediated by changes in the sensitivity of leukemic cells to retinoids. To investigate possible mechanisms of retinoid resistance, we established subclones of NB4 that are stably resistant to both tRA and 9-cisRA. Unlike the previously reported NB4.306 retinoid-resistant cells, these subclones expressed PML/RAR-alpha RNA and protein, but demonstrated altered ligand binding patterns of PML/RAR-alpha and differed in retinoid-induced gene expression. They were significantly less able to stimulate transcription of an RARE driven CAT-reporter gene on induction by tRA and showed altered DNA binding activity on a RARE. These data suggest that NB4 cells selected for resistance to retinoids demonstrate abnormal ligand binding to PML/RAR-alpha that lead to altered transcriptional activation by retinoids.

The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

Molecular genetics of cystinuria in French Canadians: identification of four novel mutations in type I patients.

Cystinuria, a hereditary disorder of cystine and dibasic amino acid reabsorption, has been classified into three subtypes on the basis of urinary excretion in obligate heterozygous parents. Thirteen cystinuric patients, identified primarily through the Quebec newborn urinary screening program, were investigated by phenotypic classification and by mutational analysis of the D2H (rBAT) gene. Mutations were identified on 7 of 25 alleles; all of these 7 mutant alleles were associated with Type I cystinuria. Four of the mutations (a large deletion, a 5'splice site mutation, a 2 bp deletion, and a nonsense mutation) have not been previously reported. These findings suggest that abnormalities in the D2H gene may account for only one subtype (Type I) of cystinuria, and that this subtype can be caused by a wide variety of population-specific mutations.

Patterns of expression of SSTR1 and SSTR2 somatostatin receptor subtypes in the hypothalamus of the adult rat: relationship to neuroendocrine function.

The neuropeptide somatostatin is the major physiological inhibitor of growth hormone secretion. With the aim of identifying the receptor subtypes through which this neuropeptide may be exerting its neuroendocrine actions in the brain, we have examined by in situ hybridization the distribution of the messenger RNA for SSTR1 and SSTR2 isoforms in the hypothalamus of adult male and female rats. Both receptor subtypes were highly expressed in the medial preoptic area, suprachiasmatic nucleus and arcuate nucleus. High SSTR1, but low SSTR2, expression was evident in the para- and periventricular nuclei as well as in the ventral premammillary nucleus. Conversely, moderate to high SSTR2, but low SSTR1, messenger RNA levels were detected in the anterior hypothalamic nucleus, ventromedial and dorsomedial nuclei and medial tuberal nucleus. Taken together, these distributional patterns conform to those of somatostatin binding sites as visualized by in vitro autoradiography, suggesting that an important proportion of SSTR1 and SSTR2 receptors in the hypothalamus are associated with the perikarya and dendrites of intrinsic neurons. The distribution of SSTR1-expressing cells within the periventricular, paraventricular and suprachiasmatic nuclei was similar to that of neurons previously reported to contain and/or express somatostatin in the brain suggesting that some of the SSTR1 receptors may correspond to autoreceptors. Within the arcuate nucleus, the distribution of SSTR1 and SSTR2 messenger RNA-expressing cells was comparable to that of neurons previously found to selectively bind somatostatin-14 within this area. Given that over one third of these cells also contain and express growth hormone-releasing factor, the present findings suggest that both of these receptor subtypes are involved in the central regulation of growth hormone-releasing factor secretion by somatostatin. Taken together, the present results suggest that SSTR1 and SSTR2 somatostatin receptor messenger RNAs are heavily expressed in those neurons containing somatostatin and/or growth hormone-releasing factor and thereby imply a role for both SSTR1 and SSTR2 somatostatin receptor subtypes in neuroendocrine regulation of growth hormone secretion in both sexes of this species.

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD).

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.

An isoenzyme study in the genus Lotus (Fabaceae) : Segregation of isoenzyme alleles in synthetic allo- and autotetraploids, and in L. corniculatus.

Segregation of the cytosolic Pgi2 locus was studied among progeny of the synthetic allotetraploid (L. japonicus × L. alpinus)(2), the synthetic autotetraploid (L. alpinus)(2), and the cultivated tetraploid species L. corniculatus L. Evidence of an original diploid duplication found within the interspecific hybrid L. japonicus × L. alpinus was also found within the synthetic allotetraploid (quadruplication of loci). Evidence suggesting quadruplication of loci was also found in the tetraploid L. corniculatus, but not in the synthetic autotetraploid (L. alpinus)(2). It is suggested that the original duplication resulted from unequal crossing-over between homoeologues and that it provides evidence that L. corniculatus is a segmental allotetraploid. Quadruplication of loci in L. corniculatus could explain previously reported distorted tetrasomic ratios for segregation of qualitative characters in this species.

An isoenzyme study in the genus Lotus (Fabaceae). Experimental protocols and genetic basis of electrophoretic phenotype.

An isoenzyme survey of some taxa in the genus Lotus (Fabaceae) was undertaken to increase the number of genetic markers available to breeders and to students of Lotus phylogeny. Twenty-one enzymes were examined using starch gel electrophoresis and nine buffer systems. Clear, consistent banding patterns were obtained for PGI, TPI, MDH, IDH (NADP), PGM, 6-PGDH, and ME. Clear but inconsistent banding patterns were obtained for FDP, G3PDH (NADP), ß-EST, LAP, MDH, DIA, and NADHDH. Phenotypes of the seven consistently resolved enzyme systems were obtained for different tissues for each of several genotypes at different stages of development. Variation in enzyme phenotypes of the same individuals under different growth conditions indicated the presence of different isozymic forms of these enzymes. Shoot tissue of plants over 6 weeks of age was found to be suitable material for further genetic studies, since phenotype for this tissue was constant despite changes in growing conditions. A formal genetic analysis of segregation and/or recombination of allozymes for the enzymes PGM, TPI, MDH, IDH, and 6-PGDH was undertaken. Isoenzyme phenotypes were examined for the diploids L. alpinus Schleich., L. burttii Sz. Borsos, L. conimbricensis Brot., L. ornithopodioides L., L. tennis Waldst. et Kit., and L. uliginosus Schkuhr; and for the diploid interspecific hybrids L. alpinus x L. conimbricensis, L. burttii x L. ornithopodioides, and L. japonicus x L. alpinus. Several new loci were identified for Lotus, namely, Idh1, Idh2, Mdh3, Pgi1, Pgi2, Tpi1, Tpi2, and 6-Pdgh1. Duplications of loci of IDH, MDH, PGI, and 6-PGDH were detected in the diploid (2n=12) interspecific hybrid L. japonicus x L. alpinus.

Evaluation of hypotheses concerning the origin of Lotus corniculatus (Fabaceae) using isoenzyme data.

An isoenzyme survey was conducted for several geographically dispersed accessions of four diploid Lotus species, L. alpinus Schleich., L. japonicus (Regel) Larsen, L. tenuis Waldst. et Kit and L. uliginosus Schkuhr, and for the tetraploid L. corniculatus L., in order to ascertain whether isoenzyme data could offer additional evidence concerning the origin of L. corniculatus. Seven enzyme systems were examined using horizontal starch gel electrophoresis. These were PGI, TPI, MDH, IDH, PGM, 6-PGDH, and ME. Lotus uliginosus had monomorphic unique alleles, that were not found within L. corniculatus, at 7 loci. These loci and alleles are: Tpi1-112, Pgm1,2-110, Pgm3-82, Mdh3-68, 6-Pgdh1-110, 6-Pgdh2-98,95, and Me2-100. Other diploid taxa contained alleles found in L. corniculatus for these and other loci. The implications of the isoenzyme data to theories on the origin of L. corniculatus are discussed.