Clinical evaluation, biochemistry and genetic polymorphism analysis for the diagnosis of lactose intolerance in a population from northeastern Brazil

OBJECTIVE: This work aimed to evaluate and correlate symptoms, biochemical blood test results and single nucleotide polymorphisms for lactose intolerance diagnosis. METHOD: A cross-sectional study was conducted in Fortaleza, Ceará, Brazil, with a total of 119 patients, 54 of whom were lactose intolerant. Clinical evaluation and biochemical blood tests were conducted after lactose ingestion and blood samples were collected for genotyping evaluation. In particular, the single nucleotide polymorphisms C>T-13910 and G>A-22018 were analyzed by restriction fragment length polymorphism/polymerase chain reaction and validated by DNA sequencing. RESULTS: Lactose-intolerant patients presented with more symptoms of flatulence (81.4%), bloating (68.5%), borborygmus (59.3%) and diarrhea (46.3%) compared with non-lactose-intolerant patients (p<0.05). We observed a significant association between the presence of the alleles T-13910 and A-22018 and the lactose-tolerant phenotype (p<0.05). After evaluation of the biochemical blood test results for lactose, we found that the most effective cutoff for glucose levels obtained for lactose malabsorbers was <15 mg/dL, presenting an area under the receiver operating characteristic curve greater than 80.3%, with satisfactory values for sensitivity and specificity. CONCLUSIONS: These data corroborate the association of these single nucleotide polymorphisms (C>T-13910 and G>A-22018) with lactose tolerance in this population and suggest clinical management for patients with lactose intolerance that considers single nucleotide polymorphism detection and a change in the biochemical blood test cutoff from <25 mg/dL to <15 mg/dL.

Análise molecular da hipolactasia primária do tipo adulto: uma nova visão do diagnóstico de um problema antigo e frequente / Molecular analysis of adult-type hypolactasia: a new view into the diagnosis of an old frequent condition

A intolerância ao leite de vaca e seus derivados acomete grande parte da população mundial. No Brasil, também se observa elevada prevalência dessa condição. A principal causa de intolerância à lactose é a Hipolactasia Primária do Tipo Adulto (HPTA), uma condi- ção determinada geneticamente e que se caracteriza pela redução da atividade da enzima lactase a partir dos primeiros anos de vida. As bases genéticas da HPTA estão relacionadas à identificação de polimorfismos de nucleotídeo único na região promotora do gene LCT (que codifica a lactase). Conforme o genótipo, haverá persistência ou não da atividade desta enzima na idade adulta. No presente artigo, são abordados aspectos clínicos e diagnósticos desta frequente condição, à luz dos conhecimentos atuais de suas bases genético-moleculares. Os autores ressaltam a importância da análise molecular da HPTA na estratégia atual de investigação diagnóstica frente a sintomas de intolerância à lactose (AU)

Intolerance to cow's milk and its derivatives affects a great part of the world's population. In Brazil, there is also a high prevalence of this condition. The main cause of lactose intolerance is primary hypolactasia (or adult-type hypolactasia ­ ATH)), a genetically determined condition characterized by reduction of lactase activity from the first years of life. The genetic basis of ATH is related to the identification of single nucleotide polymorphisms in the promoter region of the LCT gene (encoding lactase). Depending on genotype, the activity of this enzyme will persist or not into adulthood. In this article, clinical and diagnostic aspects of this condition are discussed in light of current knowledge of its molecular genetic bases. The authors emphasize the importance of molecular analysis of ATH in the current strategy of diagnostic investigation upon symptoms of lactose intolerance (AU)

Comparison of Quick Lactose Intolerance Test in duodenal biopsies of dyspeptic patients with single nucleotide polymorphism LCT-13910C>T associated with primary hypolactasia/lactase-persistence / Comparação do Teste Quick de Intolerância à Lactose em biópsias duodenais de pacientes dispépticos com polimorfismo de nucleotídeo único LCT-13910C>T associado com hipolactasia primária/lactase persistente

PURPOSE: To analyze the usefulness of Quick Lactose Intolerance Test in relation to the genetic test based on LCT-13910C>T genotypes, previously validated for clinical practice, for primary hypolactasia/lactase-persistence diagnosis. METHODS: Thirty-two dyspeptic patients that underwent upper gastrointestinal endoscopy entered the study. Two postbulbar duodenal biopsies were taken for the Quick test, and gastric antral biopsy for DNA extraction and LCT-13910C>T polymorphism analysis. DNA was also extracted from biopsies after being used in the Quick Test that was kept frozen until extraction. RESULTS: Nine patients with lactase-persistence genotype (LCT-13910CT or LCT-13910TT) had normolactasia, eleven patients with hypolactasia genotype (LCT-13910CC) had severe hypolactasia, and among twelve with mild hypolactasia, except for one that had LCT-13910CT genotype, all the others had hypolactasia genotype. The agreement between genetic test and quick test was high (p<0.0001; Kappa Index 0.92). Most of the patients that reported symptoms with lactose-containing food ingestion had severe hypolactasia (p<0.05). Amplification with good quality PCR product was also obtained with DNA extracted from biopsies previously used in the Quick Test; thus, for the future studies antral gastric biopsies for genetic test would be unnecessary. CONCLUSION: Quick test is highly sensitive and specific for hypolactasia diagnosis and indicated those patients with symptoms of lactose intolerance.

OBJETIVO: Analisar a aplicabilidade do Teste Quick de Intolerância à Lactose em relação ao teste genético baseado nos genótipos LCT-13910C>T, previamente validado para a prática clínica, para diagnóstico de má digestão primária de lactose/digestão de lactose. MÉTODOS: Trinta e dois pacientes dispépticos submetidos à endoscopia digestiva entraram no estudo. Duas biópsias duodenais pós-bulbares foram empregadas no Teste Quick, e biópsia do antro gástrico para extração de DNA e análise do polimorfismo LCT-13910C>T. DNA também foi extraído de biópsias depois de terem sido usadas no teste Quick, e conservadas congeladas. RESULTADOS: Nove pacientes com genótipo de lactase persistente (LCT-13910CT ou LCT-13910TT) tinham normolactasia, onze pacientes com genótipo de hipolactasia (LCT-13910CC) tinham hipolactasia severa, e entre doze com hipolactasia leve, com exceção de uma que tinha genótipo LCT-13910CT, todos os demais tinham genótipo de hipolactasia. A concordância entre o teste genético e o Quick Teste foi alta (p<0,0001; Índice Kappa=0,92). A maioria dos pacientes que relataram sintomas com ingestão de alimentos com lactose tinham hipolactasia severa (p<0,05). Amplificação com produto de PCR foi obtido com DNA extraído das biópsias usadas no teste Quick; portanto, nos trabalhos futuros seria desnecessário coletar biópsia do antro gástrico para o teste genético. CONCLUSÃO: O Teste Quick é altamente sensível e específico para diagnóstico de hipolactasia e indicou aqueles pacientes com sintomas de intolerância à lactose.

Diagnóstico de intolerancia a la lactosa en adultos: rendimiento comparativo de la clínica, test de hidrógeno espirado y test genético / Comparative performance of symptoms questionnaire, hydrogen test and genetic test for lactose intolerance

Background: Genetically programmed adult-type hypolactasia affects 56% of Chilean population. Ideally, diagnosis should be confirmed. Aim: To compare diagnostic yield of genetic test, hydrogen (H2) expiratory test and a validated symptomatic structured survey (SS). Material and Methods: Patients submitted to H2 test answered a historic (anamnestic) and current SS (after the ingestion of 25 g of lactose). A blood sample was obtained for determination of genetic polymorphisms C/T_13910, C/G_13907 and G/A_22018 by polymerase chain reaction. The gold standard for diagnosis of lactose intolerance (LI) was the agreement of at least two of three tests. Results: Sixty-one participants aged 39 ± 12 years (21 males), were studied. Anamnestic SS was diagnostic of LI in all cases (score > 7), while current SS detected LI in 27/61 (46%). H2 test (an increase > 15 ppm after ingestion of 25 g of lactose) showed LI in 31/61 (51%). The locus C/G_13907 showed no polymorphism and locus G/A_22018 was in complete linkage disequilibrium with C/T_13910. Genotype C/C_13910, associated to hypolactasia, was present in 30/58 (52%). According to the gold-standard, 32/61 (52.5%) patients were diagnosed as LI. Sensitivity and specificity were, respectively, 79% and 69% for current SS, 93% and 93% for H2 test and 97% and 93% for the genetic test. The last two showed a positive likelihood ratio (LR) > 10 and a negative LR < 0.1, figures within the range considered clinically useful. Conclusions: Genotype C/C_13910 is responsible for hypolactasia in this population. Anamnestic report of symptoms after milk ingestion and symptoms after lactose ingestion, are not accurate enough. H2 and genetic tests are simple and similarly accurate to diagnose lactose intolerance in adults.

Diagnosis of adult-type hypolactasia/lactase persistence: genotyping of single nucleotide polymorphism (SNP C/T-13910) is not consistent with breath test in Colombian Caribbean population / Diagnóstico de hipolactasia tipo adulto/persistência da lactase: a genotipagem do polimorfismo de um único nucleotídeo (SNP) C/T-13910 não é consistente com o teste de hidrogênio na população do Caribe Colombiano

CONTEXT: Genotyping of single nucleotide polymorphism (SNP C/T-13910) located upstream of the lactase gene is used to determine adult-type hypolactasia/lactase persistence in North-European Caucasian subjects. The applicability of this polymorphism has been studied by comparing it with the standard diagnostic methods in different populations. OBJECTIVE: To compare the lactose hydrogen breath test with the genetic test in a sample of the Colombian Caribbean population. METHODS: Lactose hydrogen breath test and genotyping of SNP C/T-13910 were applied to 128 healthy individuals (mean age 35 ± 1). A positive lactose hydrogen breath test was indicative of hypolactasia. Genotyping was done using polymerase chain reaction/restriction fragment length polymorphism. The kappa index was used to establish agreement between the two methods. RESULTS: Seventy-six subjects (59%) were lactose-maldigesters (hypolactasia) and 52 subjects (41%) were lactose-digesters (lactase persistence). The frequencies of the CC, CT and TT genotypes were 80%, 20% and 0%, respectively. Genotyping had 97% sensitivity and 46% specificity. The kappa index = 0.473 indicates moderate agreement between the genotyping of SNP C/T-13910 and the lactose hydrogen breath test. CONCLUSION: The moderate agreement indicates that the genotyping of the SNP C/T-13910 is not applicable to determine adult-type hypolactasia/lactase persistence in the population participating in this study.

CONTEXTO: A genotipagem do SNP C/T-13910 localizado corrente acima do gene da lactase é usada para determinar hipolactasia e persistência da lactase tipo adulto em indivíduos caucasianos do Norte da Europa. A aplicabilidade deste polimorfismo tem sido estudada em comparação com métodos padronizados de diagnóstico em diferentes populações. OBJETIVO: Comparar o teste de hidrogênio expirado após a ingestão de lactose com o teste genético em uma mostra da população do Caribe Colombiano. MÉTODOS: O teste de hidrogênio expirado após a ingestão de lactose e a genotipagem do SNP C/T-13910 foram aplicados em 128 sujeitos sadios (idade media 35 ± 1). O teste de hidrogênio positivo foi indicativo de hipolactasia. A genotipagem foi feita pelo método "polymerase chain reaction/restriction fragment length polymorphism". O índice Kappa foi utilizado para estabelecer a concordância entre os dois métodos. RESULTADOS: Setenta e seis indivíduos (59%) foram mau digestores da lactose (hipolactasia) e 52 outros (41%) foram digestores da lactose (persistência da lactase). As frequências dos genotipos CC, CT e TT foram 80%, 20% e 0%, respectivamente. A genotipagem mostrou 97% da sensibilidade e 46% da especificidade. O índice kappa: 0,473 indicou moderada concordância entre os dois métodos. CONCLUSÃO: A moderada concordância indica que a genotipagem do SNP C/T-13910 nao é aplicável para determinar hipolactasia tipo adulto/persistência da lactase na população estudada.

Intolerância à lactose: mudança de paradigmas com a biologia molecular / Lactose intolerance: changing paradigms due to molecular biology

Na maioria dos mamíferos a atividade da enzima lactase diminui na parede intestinal após o desmame, caracterizando a hipolactasia primária que provoca sintomas de intolerância à lactose. A intensidade dos sintomas de distensão, flatulência, dor abdominal e diarreia variam, dependendo da quantidade de lactose ingerida, e aumentam com o passar da idade. A hipolactasia é determinada geneticamente, porém uma mutação ocorreu para que fizesse parte da humanidade tolerar o leite na idade adulta. O diagnóstico é feito por teste de tolerância, empregando a lactose como desafio. Com a descoberta dos finlandeses do polimorfismo associado com a persistência da lactase, principalmente no norte da Europa, o exame genético passou a ser outra ferramenta diagnóstica mais confortável para o intolerante. No Brasil, 43 por cento dos brancos e dos mulatos têm alelo de persistência da lactase, sendo a hipolactasia mais frequente entre os negros e japoneses. Entretanto, na prática clínica indivíduos com hipolactasia podem ser orientados a consumir alguns derivados do leite e alimentos contendo lactose sem apresentar sintomas de intolerância, enquanto que outros terão que fazer restrição de lactose na dieta.

In most mammals, lactase activity declines on the intestinal wall after weaning, characterizing primary hypolactasia that provokes symptoms of lactose intolerance. The intensity of symptoms of distention, flatulence, abdominal pain and diarrhea varies, according to the amount of ingested lactose, and increases with age. Hypolactasia is genetically determined; nonetheless, a mutation occurred that had made a part of mankind tolerate milk in adulthood. Diagnosis is made by a tolerance test, using the lactose challenge. With the discovery made by the Finns of polymorphism associated with lactase persistence, mainly, in Northern Europe, the genetic test was incorporated as a more comfortable diagnostic tool for the intolerant. In Brazil, 43 percent of Caucasian and Mulatto groups have lactase persistence allele, with hipolactasia more frequently found among Blacks and Japanese. However, in clinical practice people with hypolactasia may be advised to consume certain dairy products and food containing lactose without developing intolerance symptoms, whereas others will need a lactose restriction diet.

Correlation between lactose absorption and the C/T-13910 and G/A-22018 mutations of the lactase-phlorizin hydrolase (LCT) gene in adult-type hypolactasia

The C/T-13910 mutation is the major factor responsible for the persistence of the lactase-phlorizin hydrolase (LCT) gene expression. Mutation G/A-22018 appears to be only in co-segregation with C/T-13910. The objective of the present study was to assess the presence of these two mutations in Brazilian individuals with and without lactose malabsorption diagnosed by the hydrogen breath test (HBT). Ten milk-tolerant and 10 milk-intolerant individuals underwent the HBT after oral ingestion of 50 g lactose (equivalent to 1 L of milk). Analyses for C/T-13910 and G/A-22018 mutations were performed using a PCR-based method. Primers were designed for this study based on the GenBank sequence. The CT/GA, CT/AA, and TT/AA genotypes (lactase persistence) were found in 10 individuals with negative HBT. The CC/GG genotype (lactase non-persistence) was found in 10 individuals, 9 of them with positive HBT results. There was a significant agreement between the presence of mutations in the LCT gene promoter and HBT results (kappa = -0.9, P < 0.001). The CT/AA genotype has not been described previously and seems to be related to lactase persistence. The present study showed a significant agreement between the occurrence of mutations G/A-22018 and C/T-13910 and lactose absorption in Brazilian subjects, suggesting that the molecular test used here could be proposed for the laboratory diagnosis of adult-type primary hypolactasia.

Herencia de la mala digestión de lactosa / Heredity in lactose maldigestion

Se revisan los datos publicados sobre la posibilidad de que la mala disgestión de la lactosa del tipo adulto sea de naturaleza hereditaria. Tanto la variabilidad étnica de la frecuencia de esta característica, así como los estudios en gemelos y sobre todo las investigaciones en familias, sugieren que se trata efectivamente de un problema genético. De hecho, los estudios en familias, incluyendo el realizado en México, indican que la mala digestión de lactosa se hereda en forma autosómica recesiva