BACKGROUND

Drug resistance and the gene expression profiles might discriminate the therapy outcome, and indicate the subgroup of patients with poor prognosis. In this study we analyzed the gene expression profile in correlation with the profile of ex vivo resistance to etoposide in children with acute leukemias.

METHODS

The ex vivo drug resistance profile was determined by the MTT cytotoxicity assay performed on leukemic blasts of 56 patients. Gene expression profiles were obtained from the results of hybridization of cRNA to Human Genome U133A 2.0 ologonucleotide arrays. The following analyses were performed: correlation analysis, hierarchical clustering, the assignment of location and function. Verification of data for four selected genes (MNDA, GH1, NUDT21, RHOG) was performed by quantitative real time polymerase chain reaction in the studied population and in an independent group of 54 leukemic patients.

RESULTS

Using the permutation Spearman correlation test, a set of 233 probes/209 genes was selected. The global test confirmed the significance of the correlation of gene expression profile and resistance to etoposide (p<0.001). The NUDT21 (nudix, nucleoside diphosphate linked moiety X-type, motif 21) gene showed the strongest correlation with resistance to etoposide (FDR<0.0001%).

CONCLUSIONS

Profiling of transcriptome may help in assessing the sensitivity to drugs used in chemotherapy. Resistance to etoposide is possibly associated with a change of expression of a large number of biologically important genes that influence several cellular mechanisms.

The major isoform of hGH is a polypeptide of 191 amino acids. Human GH1-43 is an amino terminal segment of hGH1-191 which comprises the first 43 amino acids. This peptide is a potent regulator of glucose homeostasis. To facilitate our understanding of the physiological regulation of hGH1-43 an assay to measure its levels in biological fluids and extracts is needed. This communication describes the development of anti-hGH1-43 monoclonal antibodies and their use in the development of an indirect competitive ELISA for the quantification of hGH1-43. Hybridomas were produced by the fusion of FOX-NY myeloma cells with spleen cells taken from a mouse immunized with hGH1-43. The hybridomas were screened for production of antibodies to hGH1-43 by antibody capture ELISA. Hybridomas which produced antibodies reactive to hGH1-43 were cloned by limiting dilution. Three monoclonal hybridomas, CCL-1, CCL-2, and CCL-3 were subsequently obtained. These hybridomas secreted antibodies that were highly reactive towards hGH1-43 but minimally reactive towards hGH1-191. The isotypes of the mAbs secreted by CCL-1, CCL-2 and CCL-3 were all IgG1 kappa as shown by isotype specific antibody capture analysis. An indirect competitive ELISA with a detection limit that ranged from 1 to 10 ng/ml was developed using mAbs from monoclonal hybridoma CCL-3. Dose-response curves for competing hGH1-191, hPRL, and hPL indicated minimal cross-reactivity of mAbs with these hormones and conversely, a high degree of specificity for hGH1-43. Dose-response curves for dilutions of human serum and pituitary extract were parallel to the standard. The availability of a sensitive assay for the measurement of hGH1-43 will help us answer questions regarding the biosynthesis, regulation of secretion, and role of hGH1-43 in the control of glucose homeostasis.

BACKGROUND

Genetic association studies aim at finding correlations between a disease state and genetic variations such as SNPs or combinations of SNPs, termed haplotypes. Some haplotypes have a particular biological meaning such as the ones derived from SNPs located in the promoters, or the ones derived from non synonymous SNPs. All these haplotypes are "subhaplotypes" because they refer only to a part of the SNPs found in the gene. Until now, subhaplotypes were directly computed from the very SNPs chosen to constitute them, without taking into account the rest of the information corresponding to the other SNPs located in the gene. In the present work, we describe an alternative approach, called the "global method", which takes into account all the SNPs known in the region and compare the efficacy of the two "direct" and "global" methods.

RESULTS

We used empirical haplotypes data sets from the GH1 promoter and the APOE gene, and 10 simulated datasets, and randomly introduced in them missing information (from 0% up to 20%) to compare the 2 methods. For each method, we used the PHASE haplotyping software since it was described to be the best. We showed that the use of the "global method" for subhaplotyping leads always to a better error rate than the classical direct haplotyping. The advantage provided by this alternative method increases with the percentage of missing genotyping data (diminution of the average error rate from 25% to less than 10%). We applied the global method software on the GRIV cohort for AIDS genetic associations and some associations previously identified through direct subhaplotyping were found to be erroneous.

CONCLUSIONS

The global method for subhaplotyping can reduce, sometimes dramatically, the error rate on patient resolutions and haplotypes frequencies. One should thus use this method in order to minimise the risk of a false interpretation in genetic studies involving subhaplotypes. In practice the global method is always more efficient than the direct method, but a combination method taking into account the level of missing information in each subject appears to be even more interesting when the level of missing information becomes larger (>10%).

A novel β-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting β-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) β-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-β-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 β-glucosidase and its applications in bioconversion and biotechnology.

Extrapituitary expression of the growth hormone (GH) gene has been reported for the immune system of various vertebrates. In the rainbow trout (Oncorhynchus mykiss), GH mRNA could be detected in several lymphoid organs and leucocytes by reverse transcriptase-polymerase chain reaction (RT-PCR). To understand the control of GH expression in the fish immune system, mRNA levels for two distinct GH genes (GH1 and GH2) in trout leucocytes isolated from peripheral blood were quantified using a real-time PCR method. Both GH mRNAs could be detected in trout leucocytes, although their levels were extremely low compared to those in pituitary cells. The levels of GH2 mRNA in leucocytes were several times higher than those of GH1, while no difference was observed between GH1 and GH2 mRNA levels in the pituitary. Administration of dibutyryl cyclic AMP and cortisol produced a significant elevation of GH mRNA levels in trout leucocytes, although the levels were unchanged by T3. GH1 and GH2 mRNA levels showed similarities in responses to those factors. The effect of cortisol on GH mRNA appears biphasic; a dose-depending elevation of GH gene expression was observed in leucocytes treated with cortisol at below 200 nM, however, cortisol had no effect at 2000 nM. Cortisol-treated leucocytes showed no significant change in the mRNA level of beta-actin or proliferative activity during the experiments. Our results thus show that, at the low levels, GH gene expression in trout leucocytes is regulated by cortisol, which has been known as a regulatory factor of GH gene expression in pituitary cells, and suggest a physiological significance of paracrine GH produced in the fish immune system.

Growth hormone (GH) is encoded by the GH gene, which may be single copy or duplicate in sheep. The two copies of the sheep GH gene (GH1/GH2-N and GH2-Z) were entirely sequenced in one 106 ewes of Sarda breed, in order to highlight sequence polymorphisms and investigate possible association between genetic variants and milk traits. Milk traits included milk yield, fat, protein, casein and lactose percentage. We evidenced 75 nucleotide changes. Transcription factor binding site prediction revealed two sequences potentially recognised by the pituitary-specific transcription factor POU1FI at the GH1/GH2-N gene, which were lost at the promoter of GH2-Z, which might explain the different tissues of expression of GH1/GH2-N (pituitary) and GH2-Z (placenta). Significant differences in milk traits were observed among genotypes at polymorphic loci only for the GH2-Z gene. Sheep with homozygote genotype ss748770547 CC had higher fat percentage (P < 0.01) than TT. SNP ss748770547 was part of a potential transcription factor binding site for C/EBP alpha (CCAAT/Enhancer Binding Protein), which is involved in the regulation of adipogenesis and adipoblast differentiation. SNP ss748770547, located in the GH2-Z gene 5' flanking region, may be a causal mutation affecting milk fat content. These findings might contribute to the knowledge of the sheep GH locus and might be useful in selection processes in sheep.

Although it is difficult to reach international agreement on the definition of growth hormone deficiency (GHD) in children and adolescents, great efforts to do so have been made during the last two decades. A somewhat limited definition of GHD is: a combination of auxological, clinical, biochemical and metabolic abnormalities caused by lack or insufficiency of GH secretion that results in a decrease in the production of GH-dependent hormones and growth factors. Its aetiology is very complex. Therefore, specific studies must be performed during different periods of childhood (neonatal, prepubertal and pubertal periods). Auxological parameters, particularly growth velocity (GV), are still considered the best clinical measures for analysing human growth. The spectacular advances in our understanding of molecular biology during the past twenty years have allowed, and will continue to allow, a more and more precise diagnosis of the molecular anomalies of human growth. This will, in turn, allow changes caused by genetic lesions to be more efficiently distinguished from those due to nutritional, organic, tumoural, psychological or traumatic causes. Our knowledge of the molecular bases of undergrowth due to a deficiency in GH has developed as a result of the localisation and characterisation of human genes which code for proteins implicated in the hormonal regulation of growth. These genes include pituitary GH (GH1), pituitary transcription factor 1 (Pit-1), the prophet of Pit-1 (PROP-1), the pituitary transcription factor LHX3, the transcription factor HESX1 and the GH-releasing hormone receptor (GHRHr). In addition, magnetic resonance imaging is the best available imaging method for the evaluation of size and structure of the pituitary and the parasellar region.

The rat provides valuable and sometimes unique models of human complex diseases. To fully exploit the rat models in biomedical research, it is important to have access to detailed knowledge of the rat genome organization as well as its relation to the human genome. Rat Chromosome 10 (RNO10) harbors several important cancer-related genes. Deletions in the proximal part of RNO10 were repeatedly found in a rat model for endometrial cancer. To identify functional and positional candidate genes in the affected region, we used radiation hybrid (RH) mapping and single- and dual-color fluorescence in situ hybridization (FISH) techniques to construct a detailed chromosomal map of the proximal part of RNO10. The regional localization of 14 genes, most of them cancer-related ( Grin2a, Gspt1, Crebbp, Gfer, Tsc2, Tpsb1, Il9r, Il4, Irf1, Csf2, Sparc, Tp53, Thra1, Gh1), and of five microsatellite markers ( D10Mit10, D10Rat42, D10Rat50, D10Rat72, and D10Rat165) was determined on RNO10. For a fifteenth gene, Ppm1b, which had previously been assigned to RNO10, the map position was corrected to RNO6q12-q13.

The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.

Isolated growth hormone deficiency (IGHD) is the commonest pituitary hormone deficiency resulting from congenital or acquired causes, although for most patients its etiology remains unknown. Among the known factors, heterozygous mutations in the growth hormone gene (GH1) lead to the autosomal dominant form of GHD, also known as type II GHD. In many cohorts this is the commonest form of congenital isolated GHD and is mainly caused by mutations that affect the correct splicing of GH-1. These mutations cause skipping of the third exon and lead to the production of a 17.5-kDa GH isoform that exerts a dominant negative effect on the secretion of the wild type GH. The identification of these mutations has clinical implications for the management of patients, as there is a well-documented correlation between the severity of the phenotype and the increased expression of the 17.5-kDa isoform. Patients with type II GHD have a variable height deficit and severity of GHD and may develop additional pituitary hormone defiencies over time, including ACTH, TSH and gonadotropin deficiencies. Therefore, their lifelong follow-up is recommended. Detailed studies on the effect of heterozygous GH1 mutations on the trafficking, secretion and action of growth hormone can elucidate their mechanism on a cellular level and may influence future treatment options for GHD type II.

Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl β-D-mannoside (4NPMan) compared with 4-nitrophenyl β-D-glucoside (4NPGlc). To investigate its selectivity for β-D-mannoside and β-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space group P212121. The (β/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with β-D-mannose in the (1)S5 skew-boat conformation. Docking of the (1)S3, (1)S5, (2)SO and (3)S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the (1)S5 and (1)S3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.

BACKGROUND

Congenital Hypopituitarism is caused by genetic and environmental factors. Over 30 genes have been implicated in isolated and/or combined pituitary hormone deficiency. The etiology remains unknown for up to 80% of the patients, but most cases have been analyzed by limited candidate gene screening. Mutations in the PROP1 gene are the most common known cause, and the frequency of mutations in this gene varies greatly by ethnicity. We designed a custom array to assess the frequency of mutations in known hypopituitarism genes and new candidates, using single molecule molecular inversion probes sequencing (smMIPS).

METHODS

We used this panel for the first systematic screening for causes of hypopituitarism in children. Molecular inversion probes were designed to capture 693 coding exons of 30 known genes and 37 candidate genes. We captured genomic DNA from 51 pediatric patients with CPHD (n = 43) or isolated GH deficiency (IGHD) (n = 8) and their parents and conducted next generation sequencing.

RESULTS

We obtained deep coverage over targeted regions and demonstrated accurate variant detection by comparison to whole-genome sequencing in a control individual. We found a dominant mutation GH1, p.R209H, in a three-generation pedigree with IGHD.

CONCLUSIONS

smMIPS is an efficient and inexpensive method to detect mutations in patients with hypopituitarism, drastically limiting the need for screening individual genes by Sanger sequencing.

β-Fructofuranosidases or invertases (EC 3.2.1.26) are enzymes that are widely used in the food industry, where fructose is preferred over sucrose, because it is sweeter and does not crystallize easily. Since Aspergillus niger GH1, an xerophilic fungus from the Mexican semi-desert, has been reported to be an invertase producer, and because of the need for new enzymes with biotechnological applications, in this work, we describe the gene and amino acid sequence of the invertase from A. niger GH1, and the use of a synthetic gene to produce the enzyme in the methylotrophic yeast Pichia pastoris. In addition, the produced invertase was characterized biochemically. The sequence of the invertase gene had a length of 1770 bp without introns, encodes a protein of 589 amino acids, and presented an identity of 93% and 97% with invertases from Aspergillus kawachi IFO 4308 and A. niger B60, respectively. A 4.2 L culture with the constructed recombinant P. pastoris strain showed an extracellular and periplasmic invertase production at 72 h induction of 498 and 3776 invertase units (U), respectively, which corresponds to 1018 U/L of culture medium. The invertase produced had an optimum pH of 5.0, optimum temperature of 60 °C, and specific activity of 3389 U/mg protein, and after storage for 96 h at 4 °C showed 93.7% of its activity. This invertase could be suitable for producing inverted sugar used in the food industry.

The types of mutations induced by oxidatively damaged products of DNA are continuously in debate. For example, some biochemical studies have proposed that guanidinohydantoin (Gh) would induce exclusively G to C mutations, while other studies have predicted a mixture of various mutations including G to C, G to T and G to A. In addition to the nature of mutations, the exact reasons of these mutations are also not properly understood. It is suggested that Gh can easily isomerize to iminoallantoin (Ia) in a pH-dependent manner and the transition becomes complete at pH>> 8. In order to understand Gh/Ia-induced mutations, we have here studied the role of the most stable tautomer of Ia in the R- and S-enantiomeric configurations in promoting mismatch base pair complexes in DNA by employing a density functional theoretical (DFT) approach. It is found that Ia can have 39 different possible tautomeric forms each in the R- and S-enantiomeric configurations, out of which the most stable tautomer would involve the deprotonation of the N1 atom and protonation of the N3 atom. The most stable tautomer of Ia can adopt three different rotameric conformations (Ia1, Ia2, and Ia3) of comparable stabilities. It is further revealed that these rotamers of Ia can interact with different bases of DNA in 88 different possible ways. However, the interaction of G with Ia3 in both the anti- and syn-conformations would be the most stable. It is further revealed that the base pairing patterns, binding energies and electronic environments of anti-Ia3:G and G:T complexes are similar. In addition to this, it is also found that the binding patterns and energies of Gh1:G and Ia3:G complexes are similar. Based on these results, it is proposed that under physiological conditions, Gh1 may be responsible for the observed G to C mutations in DNA, while in an acidic environment Ia3 may be responsible for the same mutations. This study has led to a solid foundation for further high resolution structural studies to completely unravel Ia-induced mutagenicity in DNA.

Growth hormone 1 (GH1), a pituitary hormone, plays a key role in the regulation of growth. Both excess GH1 treatment and overexpression of a GH1 transgene promote growth of salmon, but these animals exhibit physiological abnormalities in viability, fertility and metabolism, which might be related to pituitary function. However, the molecular dynamics induced in the pituitary by excess GH1 remain unknown. In this study, we performed iTRAQ proteome analysis of the amago salmon pituitary, with and without excess GH1 treatment, and found that the expression levels of proteins related to endocrine systems, metabolism, cell growth and proliferation were altered in the GH1-treated pituitary. Specifically, pituitary hormone prolactin (2.29 fold), and somatolactin α (0.14 fold) changed significantly. This result was confirmed by proteome and transcriptome analyses of pituitary from the GH1-transgenic (GH1-Tg) amago salmon. The dynamics of protein and gene expression in the pituitary of GH1-Tg amago salmon were similar to those of pituitary treated with excess GH1. Our findings suggest that not only excess GH1 hormone, but also the quantitative changes in other pituitary hormones, might be essential for the abnormal growth of amago salmon. These data will be useful in future attempts to increase the productivity of fish farming.

A novel pituitary protein "7B2" was secreted by GH1 cells. The secretion of 7B2 was increased in the presence of human GRF in a dose-responsive manner. In contrast, a somatostatin analog, SMS 201-995, revealed the inhibitory effects on the basal- and GRF-induced secretion of 7B2 at the concentration of 10(-7) M. These findings suggest that 7B2 is a secretory protein of rat GH1 cells under certain conditions.

To understand the common gene expression patterns employed by P. placenta during lignocellulose degradation, we have retrieved genome wide transcriptome datasets from NCBI GEO database and analyzed using customized analysis pipeline. We have retrieved the top differentially expressed genes and compared the common significant genes among two different growth conditions. Genes encoding for cellulolytic (<em>GH1</em>, GH3, GH5, <em>GH1</em>2, <em>GH1</em>6, GH45) and hemicellulolytic (<em>GH1</em>0, GH27, GH31, GH35, GH47, GH51, GH55, GH78, GH95) glycoside hydrolase classes were commonly up regulated among all the datasets. Fenton's reaction enzymes (iron homeostasis, reduction, hydrogen peroxide generation) were significantly expressed among all the datasets under lignocellulolytic conditions. Due to the evolutionary loss of genes coding for various lignocellulolytic enzymes (including several cellulases), P. placenta employs hemicellulolytic glycoside hydrolases and Fenton's reactions for the rapid depolymerization of plant cell wall components. Different classes of enzymes involved in aromatic compound degradation, stress responsive and detoxification mechanisms (cytochrome P450 monoxygenases) were found highly expressed in complex plant biomass substrates. We have reported the genome wide expression patterns of genes coding for information, storage and processing (KOG), tentative and predicted molecular networks involved in cellulose, hemicellulose degradation and list of significant protein-ID's commonly expressed among different lignocellulolytic growth conditions.

We investigated the promoter polymorphisms of the pituitary growth hormone gene (GH1) and exon 3 deletion polymorphism (GHRd3) in its receptor gene (GHR) in 299 angiographically proven patients with coronary artery disease (CAD) and 231 asymptomatic controls enrolled in the ongoing Indian Atherosclerosis Research Study. Real time PCR based analysis of the GHR variant showed significant association of the GHRd3 deletion allele with CAD (OR 0.48, 95% CI: 0.30-0.76, P = 0.0014) and a dominant model of inheritance (Akaike information criterion = 482). The deletion allele showed significant association with high plasma HDL-c levels (P = 0.001). Sequencing of the proximal promoter region of GH1 revealed 12 novel polymorphisms and a TAGA haplotype constituted by the functional SNPs rs2005171, rs11568828, rs2005172 and rs6171, that showed significant association with CAD alone (adjusted OR of 3.31 (95% CI = 1.33-8.29, P = 0.011) and in CAD patients with diabetes (P = 0.019). Mean standardized height was associated with three of the four haplotype-tagging SNPs in the cohort (P ≤ 0.03). Eleven of the 12 polymorphic promoter SNPs contributed to 14.7% of variation in height in females in the whole dataset (P = 0.029). CAD patients with history of stroke exhibited marginally significantly lower mean height as compared to rest of the cohort (P < 0.006). In conclusion, genetic polymorphisms in the GHR gene and its ligand, GH1, may modulate the risk of CAD in the Asian Indian population.

BACKGROUND

Mutations in the genes encoding for GHRH receptor (GHRHR) and GH (GH1) are the most common cause of familial isolated GH deficiency (IGHD). GHRHR mutations are often associated with anterior pituitary hypoplasia (APH), but this has been reported almost exclusively in children older than 8 yr. We analyzed the GHRHR and measured pituitary size in a consanguineous family with the father and three of the five siblings with IGHD.

OBJECTIVE

The aim of the study was to find the mutated gene in a family with severe IGHD.

METHODS

We sequenced the whole GHRHR coding regions and the intron-exon boundaries from peripheral DNA of the index patient. After identifying the novel mutation, we sequenced the region of interest in the other members of the family. We measured the anterior pituitary volume from magnetic resonance imaging (MRI).

RESULTS

The father and the three affected children were homozygous for a new frame-shift mutation in the coding sequence of exon 4 (corresponding to the extracellular domain of the receptor) (c.340delG) that places the downstream sequence out of frame [corrected]. The mother and two unaffected siblings were heterozygous for the mutation. Two of the affected children had MRI evidence of APH before reaching 6 yr of age.

CONCLUSIONS

We describe a new mutation in the GHRHR in a family with IGHD. The presence of frank APH before age 6 yr shows that MRI-evident reduced pituitary size can be present in GHRHR mutations even in children younger than 8 yr of age.

BACKGROUND

Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner.

OBJECTIVE

Two siblings born from nonconsanguineous healthy parents exhibited IGHD. To elucidate the cause, GH1 in all family members was analysed.

RESULTS

Two novel mutations in GH1, a point mutation in intron 3 and a 16-bp deletion in exon 3, were identified by sequence analyses. The intronic mutation was present in both siblings and was predicted to cause aberrant splicing. The deletion was present in one of the siblings as well as the mother with normal stature and was predicted to cause rapid degradation of mRNA through nonsense-mediated mRNA decay. The point mutation was not identified in the parents' peripheral blood DNA; however, it was detected in the DNA extracted from the father's sperms. As a trace of the mutant allele was detected in the peripheral blood of the father using PCR-RFLP, the mutation is likely to have occurred de novo at an early developmental stage before differentiation of somatic cells and germline cells.

CONCLUSIONS

This is the first report of mosaicism for a mutation in GH1 in a family with IGHD. It is clear that the intronic mutation plays a dominant role in the pathogenesis of IGHD in this family, as one of the siblings who had only the point mutation was affected. On the other hand, the other sibling was a compound heterozygote for the point mutation and the 16-bp deletion and it may be arguable whether IGHD in this patient should be regarded as autosomal dominant or recessive.

The aim of the present study was to identify and characterize polymorphisms within the 5' flanking region, first exon and part of first intron of the bovine growth hormone gene among different beef cattle breeds: Nelore (n = 25), Simmental (n = 39), Simbrasil (n = 24), Simmental x Nelore (n = 30), Canchim x Nelore (n = 30) and Angus x Nelore (n = 30). Two DNA fragments (GH1, 464 bp and GH2, 453 bp) were amplified by polymerase chain reaction and then used for polymorphism identification by SSCP. Within the GH1 fragment, five polymorphisms were identified, corresponding to three different alleles: GH1.1, GH1.2 and GH1.3 (GenBank: AY662648, AY662649 and AY662650, respectively). These allele sequences were aligned and compared with bovine GH gene nucleotide sequence (GenBank: M57764 and AF118837), resulting in the identification of five insertion/deletions (INDELs) and five single nucleotide polymorphisms (SNPs). In the GH2 fragment two alleles were identified, GH2.1 and GH2.2 (GenBank: AY662651 and AY662652, respectively). The allele sequences were compared with GenBank sequences (M57764, AF007750 and AH009106) and three INDELs and four SNPs were identified. In conclusion, we were able to identify six new polymorphisms of the bovine GH gene (one INDEL and five SNPs), which can be used as molecular markers in genetic studies.