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Variations in the Promoter Region of the Glutaminase Gene and the Development of Hepatic Encephalopathy in Patients With Cirrhosis: A Cohort Study

Manuel Romero-Gómez, MD, PhD; María Jover, PhD; José A. Del Campo, PhD; José L. Royo, PhD; Elena Hoyas, MD; José J. Galán, PhD; Carmina Montoliu, PhD; Eugenia Baccaro, MD; Mónica Guevara, MD, PhD; Juan Córdoba, MD, PhD; Germán Soriano, MD, PhD; José M. Navarro, MD; Carmen Martínez-Sierra, MD, PhD; Lourdes Grande, MD, PhD; Antonio Galindo, MD, PhD; Emilia Mira, PhD; Santos Mañes, PhD; and Agustín Ruiz, MD, PhD
[+] Article, Author, and Disclosure Information

From Hospital Universitario de Valme, Universidad de Sevilla, and Neocodex, Sevilla; Hospital Clínico de Valencia, Valencia; Hospital Clinic, Hospital Vall d'Hebron, and Hospital de la Santa Creu i Sant Pau, Barcelona; Hospital Costa del Sol, Marbella, Málaga; Hospital Puerta del Mar, Cádiz; and Centro Nacional de Biotecnología, Madrid, Spain.

Note: Drs. Jover and Del Campo contributed equally to this work.

Acknowledgment: The authors thank Peter R. Turner for editorial assistance.

Grant Support: By the Spanish Ministry of Health (Instituto de Salud Carlos III, grants PI040384 and PI070425).

Potential Conflicts of Interest: Disclosures can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M10-0301.

Requests for Single Reprints: Manuel Romero-Gómez, MD, PhD, Unit for the Clinical Management of Digestive Diseases and CIBERehd, Hospital Universitario de Valme, Universidad de Sevilla, 41014 Sevilla, Spain; e-mail, mromerogomez@us.es.

Reproducible Research Statement:Study protocol and statistical code: Available from Dr. Romero-Gómez (e-mail, mromerogomez@us.es). Data set: Available from Dr. Romero-Gómez after formal written agreement.

Current Author Addresses: Drs. Romero-Gómez, Jover, Del Campo, Hoyas, Grande, and Galindo: Unit for the Clinical Management of Digestive Diseases and CIBER de Enfermedades Hepáticas y Digestivas (CIBERehd), Hospital Universitario de Valme, Avenida de Bellavista s/n, 41014 Sevilla, Spain.

Dr. Royo: Centro Andaluz de Biología del Desarrollo, Spanish Research Council (CSIC)–University Pablo de Olavide, 41013 Sevilla, Spain.

Drs. Galán and Ruiz: Department of Structural Genomics, Neocodex SL, Avenida Charles Darwin s/n, Parque Científico y Tecnológico, Isla de la Cartuja, 41092 Sevilla, Spain.

Dr. Montoliu: Fundacion Investigacion Hospital Clinico de Valencia, Avenida Blasco Ibañez 17, 46010 Valencia, Spain.

Drs. Baccaro and Guevara: Clinical Research, Liver Unit Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, CIBERehd, Villarroel 170, Unidad de Hepatología (planta 3 entre esc 7 y 9), 08036 Barcelona, Spain.

Dr. Córdoba: Universidad Autónoma de Barcelona, Hospital Vall d'Hebron, Paseo Vall d'Hebron 119, 08035 Barcelona, Spain.

Dr. Soriano: Department of Gastroenterology, CIBERehd, Hospital de la Santa Creu i Sant Pau, Mas Casanovas 90, 08041 Barcelona, Spain.

Dr. Navarro: Gastroenterology and Hepatology Unit, Hospital Costa del Sol, Marbella, 29602 Málaga, Spain.

Dr. Martínez-Sierra: Gastroenterology Unit, Hospitales Universitarios Virgen del Rocio, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain.

Drs. Mira and Mañes: National Center of Biotechnology/CSIC, Department of Immunology and Oncology, Darwin 3, Campus Cantoblanco, 28049 Madrid, Spain.

Author Contributions: Conception and design: M. Romero-Gómez, J.L. Royo, A. Ruiz.

Analysis and interpretation of the data: M. Romero-Gómez, M. Jover, J.A. Del Campo, J.L. Royo, E. Mira, S. Mañes, A. Ruiz.

Drafting of the article: M. Romero-Gómez, A. Ruiz.

Critical revision of the article for important intellectual content: M. Romero-Gómez, M. Jover, J.A. Del Campo, J.L. Royo, J.J. Galán, C. Montoliu, E. Baccaro, M. Guevara, J. Córdoba, G. Soriano, J.M. Navarro, C. Martínez-Sierra, L. Grande, A. Galindo, E. Mira, S. Mañes, A. Ruiz.

Final approval of the article: M. Romero-Gómez, M. Jover, J.A. Del Campo, J.L. Royo, E. Hoyas, J.J. Galán, C. Montoliu, E. Baccaro, M. Guevara, J. Córdoba, G. Soriano, J.M. Navarro, C. Martínez-Sierra, A. Galindo, E. Mira, S. Mañes, A. Ruiz.

Provision of study materials or patients: M. Romero-Gómez, M. Jover, J. A. Del Campo, E. Hoyas, M. Guevara, J. Córdoba, G. Soriano, C. Martínez-Sierra, L. Grande, A. Galindo, S. Mañes.

Statistical expertise: M. Romero-Gómez, A. Ruiz.

Obtaining of funding: M. Romero-Gómez.

Administrative, technical, or logistic support: M. Romero-Gómez, M. Jover, J.A. Del Campo, J.L. Royo, J.J. Galán.

Collection and assembly of data: M. Romero-Gómez, M. Jover, E. Hoyas, J.J. Galán, A. Ruiz.

Ann Intern Med. 2010;153(5):281-288. doi:10.7326/0003-4819-153-5-201009070-00002
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Background: Hepatic encephalopathy is a major complication of cirrhosis and is associated with a poor prognosis.

Objective: To identify mutations in the gene sequence for glutaminase in humans that could be responsible for the development of hepatic encephalopathy in patients with cirrhosis.

Design: Cohort study.

Setting: Outpatient clinics in 6 Spanish hospitals.

Patients: 109 consecutive patients with cirrhosis in the estimation cohort, 177 patients in the validation cohort, and 107 healthy control participants.

Measurements: Patients were followed every 3 or 6 months until the development of hepatic encephalopathy or liver transplantation, death, or the end of the study.

Results: The genetic analyses showed that glutaminase TACC and CACC haplotypes were linked to the risk for overt hepatic encephalopathy. Mutation scanning of the glutaminase gene identified a section in the promoter region where base pairs were repeated (a microsatellite). Over a mean follow-up of 29.6 months, hepatic encephalopathy occurred in 28 patients (25.7%) in the estimation cohort. Multivariable Cox models were used to determine the following independent predictors: Child–Turcotte–Pugh stage (hazard ratio [HR], 1.6 [95% CI, 1.29 to 1.98]; P = 0.001), minimal hepatic encephalopathy (HR, 3.17 [CI, 1.42 to 7.09]; P = 0.006), and having 2 long alleles of the microsatellite (HR, 3.12 [CI, 1.39 to 7.02]; P = 0.006). The association between 2 long alleles of the microsatellite and overt hepatic encephalopathy was confirmed in a validation cohort (HR, 2.1 [CI, 1.17 to 3.79]; P = 0.012). Functional studies showed higher luciferase activity in cells transfected with the long form of the microsatellite, which suggests that the long microsatellite enhances glutaminase transcriptional activity.

Limitation: Other genes and allelic variants might be involved in the clinical expression of hepatic encephalopathy.

Conclusion: This study identifies a genetic factor that is associated with development of hepatic encephalopathy in patients with cirrhosis.

Primary Funding Source: Instituto de Salud Carlos III, Spanish Ministry of Health.


Grahic Jump Location
Appendix Figure 1.
Schematic representation of the constructs used to determine the functional role of the different alleles.

Clones included 1.5-kb material upstream of the glutaminase transcription start site (+1 in bold) together with the 5′ untranslated region (underlined). To determine potential translational defects associated with the microsatellite (italic), the first 6 glutaminase amino acids were also included in the constructs (in red) and conveniently cloned in-frame to the luciferase gene (in blue).

Grahic Jump Location
Grahic Jump Location
Figure 1.
Detection of a microsatellite in the 5′ untranslated region of the glutaminase gene.

The 2 panels are examples of the microsatellite region in 2 patients. The dotted line peaks represent the microsatellite alleles in each patient. The solid line represents internal markers to calculate the exact size of the alleles. In this example, both patients shared 1 of their 2 alleles, which were the same size. Dye signal was calculated by using a CEQ8000 device (Beckman Coulter, Fullerton, California) by capillary electrophoresis.

Grahic Jump Location
Grahic Jump Location
Appendix Figure 2.
Frequency distribution of microsatellite alleles in study participants (768 chromosomes).
Grahic Jump Location
Grahic Jump Location
Figure 2.
Development of overt hepatic encephalopathy over time, by microsatellite length group.

Short alleles are those with fewer than 14 GCA tandem repeats, and long alleles are those with 14 or more GCA tandem repeats. Results of the log-rank test for the estimation cohort, 8.41 (P = 0.004); for the validation cohort, 9.21 (P = 0.002).

Grahic Jump Location
Grahic Jump Location
Figure 3.
Ratio of Renilla luciferase activity to Drosophila luciferase activity measured 24, 48, and 72 h after transfection.

P values are from overall Kruskal–Wallis tests comparing the ratios of Renilla luciferase activity with Drosophila luciferase activity by microsatellite length (8 vs. 14 vs. 29 GCA repeats). Error bars represent 95% CIs.

Grahic Jump Location




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Effective Hepatitis-E vaccine -urgently needed
Posted on October 7, 2010
Conflict of Interest: None Declared

The article by Romero-Gomez M et al is invaluable (1). Chronic liver disease (CLD) is increasing worldwide especially in developing and least developed countries. Extrapolated statistics showed prevalence of CLD in India (1,566,280) and other developing countries were higher than USA (431,846), UK (88,633) and other developed countries and might be under reflecting the true disease burden [2]. The commonest causes of acute decompensation of CLD are hypovolaemia (GI bleed), alcohol, sepsis, drugs, electrolyte disturbance and super infection with hepatotropic viruses like HAV, HEV.

HEV is responsible for epidemics and sporadic hepatitis in southeast and central Asia, the Middle East, parts of Africa and Mexico. The genotypes 1 and 2 infect humans through contaminated drinking water and food and commonly affect young adults and mortality rate is usually 0.5- 4.0%, but may be high (20%) among pregnant women in 3rd trimester due to fulminant hepatitis [3]. Genotypes 3 and 4 infect animals and zoonotic transmission from pigs, wild boars and deer, either food-borne or otherwise causing hepatitis in elderly [4].Super infection with HEV in CLD causes severe liver decompensation with hepatic encephalopathy and renal failure and patients with CLD were at increased risk of developing hepatitis-E (44%) [5].

Poor sanitation and water contamination, poor awareness of health and hygiene, possible zoonotic transmission and high increasing prevalence of CLD constitute a significant HEV related mortality and morbidity in developing countries and lack of an effective HEV vaccine might be a serious threat if gene responsible for overt hepatic encephalopathy is prevalent [1].


1. Romero-G?mez M et al: Variations in the Promoter Region of the Glutaminase Gene and the Development of Hepatic Encephalopathy in Patients with Cirrhosis. Ann Intern Med September 7, 2010 vol. 153 no. 5 281-28.

2. Prevalence and Incidence of Chronic liver disease - www.wrongDiagnosis.com

3. WHO | Hepatitis E: www.who.int ? ... ? Media centre ? Fact sheets

4. Aggarwal R, Naik S. Epidemiology of hepatitis E: current status.J Gastroenterol Hepatol. 2009 Sep; 24(9):1484-93. Epub 2009 Aug 3.

5. Ramachandran J et al.Hepatitis E super infection produces severe decompensation in patients with chronic liver disease. J Gastroenterol Hepatol. 2004 Feb; 19(2):134-8.

Conflict of Interest:

None declared

Hepatic and neurologic manifestations : clinics today
Posted on November 3, 2010
Conflict of Interest: None Declared

Recent introduction of subthelomeric rearrangements methodics : multiplex ligation probe amplification ( MLPA ) has deserved a useful tool , since the introduction of the method in the '90s , this tool has been implemented for the detection of losses or gains in in genetic cryptic exploration. A very interesting prospective area , would be derived from the possible correlation between unexplained encephalopathies in hepatic and non- hepatic patients , which may correlate with cryptic vascular malformations , such porto-systemic shunts in hepatic encephalopathy

Conflict of Interest:

None declared

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