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After the description of lactose intolerance, it was discovered that adult lactose maldigestion is a recessive (lactase nonpersistence, LNP), whereas the ability to digest lactose is a dominant genetic trait (lactase persistence, LP).1, 2 Distributions depend on ethnic and geographic factors, such that the majority (about 65%) is LNP.3 LNP status generally increases towards the equator and eastward. The new worlds (North and South America, Australia) are variably distributed due to historical population migrations.
Two features of lactase dichotomy have interested workers in the field. In medicine and nutrition, the clinical contribution of lactose maldigestion to the development of gastrointestinal symptoms fuelled research. As a result, to alleviate symptoms, there is importance attributed to alter dietary use of dairy foods.4 The other aspect is medical epidemiology, and partly relates the global distribution of LP/LNP with risks of several (possibly many) diseases.5
Research interests to date relied mainly on indirect tests of lactose digestion. These tests were generally correlated with intestinal biopsy measurements of lactase,6, 7 but for clinical or epidemiological studies this method is not practical. Based on previous comparisons of indirect tests, current methodology employs mainly the lactose breath hydrogen test (LBHT) and secondly the lactose tolerance tests (LTT).8, 9 The latter measures the presence of intestinal lactase and the ability of the person to split lactose into glucose and galactose. A positive rise of glucose (>1.1–1.4 g/L within 2 h) is consistent with LP status.9 The breath test measures bacterial metabolism of lactose, which spills into the lower intestine when inadequately metabolised. Hydrogen produced by bacterial metabolism enters the lung, is exhaled and is measured in the breath.10 A rise of hydrogen (≥20 parts per million, ppm), is consistent with LNP status. There is only a moderate agreement between these two indirect tests.8, 11, 12
The genetics of lactase has interested different groups, and knowledge has advanced considerably. Control of this gene on chromosome 2q21 has been identified with several haplotypes. The main site is in intron 13 of the minichromosome maintenance 6 (MCM 6) gene (reviewed in 2). The first gene to control transcription of lactase, the C/T-13910 was identified by Enattah et al. and is noted largely in north Europeans and their descendants.13 This polymorphism was correlated with intestinal biopsies.14 A second polymorphism G/A-22018 has also been described but in Europeans may not have independent effects. It, however, may be the main polymorphism controlling lactase in northern Chinese.15, 16 In north Europeans, C/C genotypes are homozygous maldigesters with less than 10 U/g intestinal lactase (LNP), T/T genotypes are normal digesters with normal intestinal lactase levels (LP), and C/T genotypes are normal digesters with intermediate intestinal enzyme levels (LP).
Other polymorphisms related to the European type have been identified in east Africa and the Middle East.17, 18 The presence of these or other single nuclear polymorphisms could interfere with currently used polymerase chain reaction (PCR)-based kits measuring the C/T-13910 polymorphism. With increasing variety of LP polymorphisms possibly contributing to populations towards the south of Europe, it was suggested that current kits be used with caution for genetic identification in non-Northern Europeans because diagnostic precision may be lost (loss of sensitivity and specificity).19
The objectives of this systematic review were to evaluate studies comparing the LBHT or LTT with north-European genetic polymorphisms (currently the only commercially available test kits) for accuracy. Possible confounders that might reduce test accuracy are evaluated. The diagnostic precision of the G/A polymorphism is also evaluated from available literature.
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This report shows that LBHT is sensitive, specific and has a high DOR for the C/T-13910 European polymorphism. Heterogeneity is partly explained by dose of lactose, inclusion of children and perhaps latitude of study centres. While there were few studies with LTT, accuracy of this test was also good. Some caution may be needed to accept conclusions due to presence of publication bias.
An early comparison of indirect tests with intestinal lactase showed the LBHT to be superior to the LTT.8 However, intestinal lactase declines in a spotty fashion, especially in young subjects, making site of biopsy relevant.54 Also agreement is modest-to-moderate between LBHT and LTT8, 11, 12, 55 with overlapping modifiers of each test reducing reliability (Supporting Information, Table S3), questioning accuracy to predict geno/phenotypes. As such, reassessment of these tests with genetics is timely.
There are unique aspects to these tests as they are not used for classical disease detection. Positive and negative outcomes may be confusing as outlined in the introduction. When tests are applied to genetics of lactase, a dichotomous outcome is compared with three genotypes. Heterozygotes of lactase are generally digesters. The “sensitivity” of LBHT in the C/T-13910 refers to C/C genotype, and specificity for C/T and T/T alleles. In order to keep this pattern the LTT sensitivity is the negative test.
Secondly, LBHT measures bacterial and LTT, human response. Lactase does not increase after LNP status is achieved.56 However, continued ingestion of lactose by LNP subjects could convert a positive LBHT to negative, through bacterial adaptation.57 The impact of this process on testing LNP people has not been fully explored. Allelic predictions could be skewed by unrecognised adaptation, leading to lower sensitivity, if dairy food consumption continues.5 The LTT may be free of this feature, but has not been studied.
Thirdly, response to LBHT can be modified by contribution of methane gas. Hydrogen is converted to methane in an unknown portion of populations58, 59 and could result in apparent failure to produce hydrogen, increasing false negative outcomes also.40
The DOR is best applied when threshold effects contribute to heterogeneity,23, 24 but is independent of the above test features. The open-ended scale may be less sensitive to small variations in its dependent components. This is exemplified by different outcomes of meta-regression and sub-group analysis of lactose load with the LBHT. While overall DOR was nonsignificantly affected by altered doses (despite a 43% change), sub-group analysis did show expected improvement in sensitivity and specificity. There was also improvement when studies with children (age ≤ 17) were excluded. This improved the DOR by about 63%. LNP children have different rates of lactase loss, depending on ethnicity and race.2 Statistical significance may have been difficult to reach because of limited number of studies available (frequent in meta-analyses).
Clinical heterogeneity was also evaluated by a summary ROC curve. The studies by Mottes et al.39 and to a lesser extent Waud et al.40 contributed most to heterogeneity.
Mottes et al. included children and adult relatives for genetic comparisons. Our calculated sensitivity was low but specificity was higher. The authors looked for other polymorphisms to account for poor outcome of LBHT with genotypes other than C/C, but did not find any controlling LP status. A number of test negative, C/C subjects were noted also, possibly reflecting adaptation as discussed above. The study by Waud et al. evaluated genetics and lactose “sensitivity”. Sensitivity was 100% for genotype C/C but sensitivity and specificity were poor for other genotypes. Moreover, measuring methane gas added to predicting symptoms, although the point may be controversial.60, 61The explanation for altered effects in C/T and T/T genotypes is not clear, but this study defined lactose “sensitivity” as comparator rather than digestion with genetics and and included an unknown number of children. Also positive outcome of the test was defined as a rise above the nadir in the study period. Both these definitions require validation and may have had some effects on results.
The LTT also accurately predicts genotype for LP/LNP status, although, it is based on few publications. These results nevertheless, conflict with earlier findings.8
From a clinical perspective, current practices are confirmed. A 50 g lactose load is more sensitive for genotype, while a 25 g (more physiological) indicate specific lactose sensitivity. Alternatively, failure to elicit symptoms could convince patients not to exclude dairy foods, which may have nutritional consequences.62
The second variable affecting results is the inclusion of children. Although, we used a dichotomous outcome, two of the studies39, 42 included large number of cases. Therefore, in predicting genotype, the proportion of children in a study may sway expectations in outcome.
Interests in lactase distributions also involve the evolution of the dominant polymorphisms for LP status worldwide2, 3, 16–19, 50, 52 and relationships with disease risks.5, 63 Population migrations are a putative determinant of LP status and there is a distinct north to south decreasing distribution in Europe.2, 3, 19 Also migrations to new worlds in the last 5–6 centuries emanated from north-western Europe, making early evolutionary events there, epidemiologically relevant. Many diseases including cancers (breast, prostate, colon ovary, and others) and inflammatory bowel disease are noted to have north south reduction of risks (putatively related to sunshine and vitamin D).64–66 Coincidentally, these changes are also observed with lactase status5 and have now been linked, but mainly in Europe.63 This analysis may give some insight to the extent of the CT-13910 impact on LP status and may suggest presence of other polymorphisms without availability of specific kits. As suggested,19 the site of study (defined by latitude) did affect outcome with the current kit. However, either sensitivity (biased perhaps in Europe by LBHT and microbial effects) or specificity (interference by other polymorphisms perhaps) in overall studies was affected, but not both. Frequent, alternate polymorphisms contributing to outcome might have affected both.
Limitations include a lack of power, due to paucity of reports. Intestinal diseases can cause secondary lactose maldigestion with confounding. However, only one study included a small number of patients with these diseases.42 Also analysis does not consider ageing, which may increase breath hydrogen.67 Finally, there is publication bias, but there is a dissenting opinion, whether this should be considered in meta-analyses of diagnostic tests.68 Nevertheless, these findings prompt some caution in interpretation of results. In summary, the LBHT accurately reflects LP/LNP genotype. The DOR may be a blunt instrument with genetic tests and may be more useful in detecting large population differences. Latitude as a possible co marker for genetic variation has some impact on LBHT outcome. Explanations for this effect await further developments. While in this analysis the LTT appears as accurate for predicting genotypes, it is based on limited number of small studies and requires additional verification.