Human growth hormone (GH1) gene polymorphism map in a normal-statured adult population

OBJECTIVE: GH1 gene presents a complex map of single nucleotide polymorphisms (SNPs) in the entire promoter, coding and noncoding regions. The aim of the study was to establish the complete map of GH1 gene SNPs in our control normal population and to analyse its association with adult height. DESIGN, SUBJECTS AND MEASUREMENTS: A systematic GH1 gene analysis was designed in a control population of 307 adults of both sexes with height normally distributed within normal range for the same population: −2 standard deviation scores (SDS) to +2 SDS. An analysis was performed on individual and combined genotype associations with adult height. RESULTS: Twenty-five SNPs presented a frequency over 1%: 11 in the promoter (P1 to P11), three in the 5′UTR region (P12 to P14), one in exon 1 (P15), three in intron 1 (P16 to P18), two in intron 2 (P19 and P20), two in exon 4 (P21 and P22) and three in intron 4 (P23 to P25). Twenty-nine additional changes with frequencies under 1% were found in 29 subjects. P8, P19, P20 and P25 had not been previously described. P6, P12, P17 and P25 accounted for 6·2% of the variation in adult height (P = 0·0007) in this population with genotypes A/G at P6, G/G at P6 and A/G at P12 decreasing height SDS (−0·063 ± 0·031, −0·693 ± 0·350 and −0·489 ± 0·265, Mean ± SE) and genotypes A/T at P17 and T/G at P25 increasing height SDS (+1·094 ± 0·456 and +1·184 ± 0·432). CONCLUSIONS: This study established the GH1 gene sequence variation map in a normal adult height control population confirming the high density of SNPs in a relatively small gene. Our study shows that the more frequent SNPs did not significantly contribute to height determination, while only one promoter and two intronic SNPs contributed significantly to it. Studies in larger populations will have to confirm the associations and in vitro functional studies will elucidate the mechanisms involved. Systematic GH1 gene analysis in patients with growth delay and suspected GH deficiency/insufficiency will clarify whether different SNP frequencies and/or the presence of different sequence changes may be associated with phenotypes in them

Caracterització molecular del gen GH1 en poblacions control i patològiques, patrons d'expressió en leucòcits de sang perifèrica i estudi funcional de proteïnes mutants detectades en pacients

[cat] El retard de creixement pot ser secundari en un percentatge indeterminat de pacients a un problema genètic. En aquest treball s'analitza el gen de l'hormona de creixement (GH1) i s'estudien els canvis en la seqüència que produeixin un ARN missatger anòmal o una proteïna que no sigui funcional. S'ha estudiat el mapa polimòrfic del gen GH1 en una població control (n=307), per definir el mapa de SNPs. Alhora s'ha buscat un model en el qual poder estudiar l'expressió d'aquest gen. S'ha aïllat l'ARN de leucòcits de sang perifèrica tant de pacients com de controls, i s'ha amplificat el missatger de GH1. També s'ha posat a punt un model cel·lular en el qual poder estudiar la bioactivitat de les proteïnes recombinants portadores dels canvis detectats en pacients (Phe25Tyr i Val173Leu). Les conclusions del treball han estat les següents: 1. Els nostres resultats han confirmat i ampliat la descripció de la variabilitat estructural del gen GH1. Han permès descriure el mapa complet per primera vegada, en una població adulta amb talla normal, compresa entre -2 i +2 SDS (n=307). 2. En les poblacions amb retard crònic de creixement (n=728) s'han detectat en un 11% de pacients canvis en la seqüència del gen GH1 diferents dels SNPs del mapa control. Entre els canvis, l'11,3% correspon a canvis puntuals d'un aminoàcid (1% del total de pacients índex). 3. L'expressió de GH1 en leucòcits de controls no és constitutiva ni constant en quant a les variants de splicing observades. Aquesta metodologia no permet detectar de forma fiable mutacions en pacients. En canvi, ens ha permès confirmar l'expressió de mutants com en el cas d'una substitució predictora de canvi d'aminoàcid, així com descartar o confirmar anomalies de splicing en al·lels portadors de canvis intrònics. 4. La utilització d'eines informàtiques ha permès realitzar una aproximació teòrica a l'efecte que dos canvis d'aminoàcid (Phe25Tyr i Val173Leu) a la proteïna GH tenen sobre la seva estructura i interacció amb GHR. L'anàlisi d'homologia de les proteïnes mutants amb les dels altres gens del clúster de la GH ha permès concloure que la substitució Phe25Tyr es pot haver generat per recombinació gènica amb el gen GH2, mentre que no és el cas per a la proteïna amb el canvi Val173Leu. 5. Les proteïnes recombinants obtingudes al laboratori (WT i mutants) han estat aptes per al seu estudi in vitro (inmunoreactivitat, proliferació cel·lular i bioactivitat). 6. Les GHs mutants Phe25Tyr i Val173Leu presenten una inmunoactivitat i una bioactivitat disminuïdes. Els models cel·lulars de les línies HepG2 i C-28/I 2 no han permès obtenir resultats, mentre que el model de cultiu primari de condròcits fetals humans ha permès detectar que les dues GHs mutants estimulen amb menor intensitat la transcripció de IGF1 mentre que provoquen un augment en l'expressió de GHR i IGF1R. 7. El canvi d'aminoàcid Phe25Tyr incorporat en un al·lel de GH1 portat en heterozigosi per dues famílies no relacionades es manifesta com a retard de creixement durant la infantesa que s'acompanya de resposta deficitària de secreció de GH i disminució de IGF1. La resposta al tractament amb GH és adequada i permet recomanar el tractament fins al final del creixement. El canvi Val173Leu detectat en una pacient amb talla baixa i RIUC, sense dèficit aparent de GH, permet suggerir que la detecció precoç de mutacions al gen GH1 quan la secreció aparenta ser normal, permetria un tractament més precoç que hagués millorat el seu pronòstic de talla final. L'anàlisi dels haplotips complets de GH1 permet interpretar l'efecte de la combinació de mutacions a la proteïna amb els genotips per als SNPs.[eng] TITLE OF THE THESIS: "MOLECULAR CARACTERITZATION OF GH1 GENE IN CONTROL AND PATHOLOGIC POPULATIONS, EXPRESSION PATTERNS IN PERIPHERAL BLOOD LEUCOCYTES AND FUNCTIONAL STUDY OF MUTANT PROTEINS DETECTED IN PATIENTS" SUMMARY: The growth delay can be a secondary factor in front of a genetic problem in an indeterminate percentage of patients. In this work, it is analyzed the gene of growth hormone (GH1) and it is studied the changes in the sequence that produce an anomalous messenger ARN or a protein that is not functional. The polymorphic map of gene GH1 has been studied in a control population (n=307), to define the map of SNPs. At the same time, a model has been looked for, in which it could be possible to study the expression of this gene. The ARN of leucocytes of peripheral blood of patients and controls has been isolated, and the GH1 messenger has been amplified. Moreover, we have worked in a cellular model that allows us to study the bioactivity of recombinant proteins carrying the changes detected in patients (Phe25Tyr and Val173Leu). The conclusions of the work have been the following ones: 1. Our results have confirmed and extended the description of the structural variability of gene GH1. They have allowed us to describe the complete map for the first time, in an adult population with normal stature, between -2 and +2 SDS (n=307). 2. In the populations with chronic growth delay (n=728) in an 11% of the patients, changes have been detected in the sequence of gene GH1 different from the SNPs of the map control. Within these changes, 11.3% correspond to precise changes in an amino acid (1% of the total of index patients). 3. The expression of GH1 in leukocytes of controls is neither constitutive nor constant as far as the observed variants of splicing. This methodology does not allow detecting without doubt the mutations in patients. However, it has allowed us to confirm the expression of mutants as in the case of a predicting substitution of change in amino acid, as well as discarding or confirming anomalies of splicing in carrying alleles of intronic changes. 4. The use of computer tools has allowed us to make a theoretical approach to the effect that two changes in amino acid (Phe25Tyr and Val173Leu) in protein GH have on their structure and interaction with GHR. The homology analysis of mutant proteins with those of the other genes of the cluster of the GH has allowed us to conclude that the Phe25Tyr substitution can have been generated by genic recombination with gene GH2, whereas it is not the case for the protein with the Val173Leu change. 5. The recombinant proteins obtained in the laboratory (WT and mutants) have been suitable for their study in vitro (inmunoreactivity, cellular proliferation and bioactivity). 6. The mutant GHs Phe25Tyr and Val173Leu show a diminished inmunoactivity and bioactivity. The cellular models of lines HepG2 and C-28/I2 have not allowed us to obtain results; in contrast, the model of primary culture of human foetal chondrocytes has allowed us to detect that the two mutant GHs stimulate with smaller intensity the IGF1 transcription and they cause an increase in the expression of GHR and IGF1R. 7. The change of Phe25Tyr amino acid incorporated in one allele of GH1 taken to heterozygosity by two non-related families is shown as growth delay during childhood accompanied by deficiency secretion answer of GH and diminution of IGF1. The response to the treatment with GH is suitable and allows us to recommend the treatment until the end of the growth. The Val173Leu change detected in a patient with short stature and RIUC, without apparent GH deficiency, allows us to suggest that precocious detection of mutations in gene GH1 when the secretion pretends to be normal, would allow a precocious treatment that would have improved its prognosis of final stature. The analysis of the complete haplotypes of GH1 allows us to interpret the effect of the combination of mutations in the protein with the genotypes for the SNPs

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially