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Decline of Lactase Activity and C/T-13910 Variant in Sardinian Childhood

Schirru, Enrico*; Corona, Valeria; Usai-Satta, Paolo; Scarpa, Maria; Cucca, Francesco§; De Virgiliis, Stefano*; Rossino, Rossano*; Frau, Fulvia; Macis, Maria Doloretta*; Jores, Rita-Désirée; Congia, Mauro||

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Journal of Pediatric Gastroenterology and Nutrition: October 2007 - Volume 45 - Issue 4 - p 503-506
doi: 10.1097/MPG.0b013e31805b5899
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In the majority of human populations, a gradual decline of lactase activity is observed during childhood. The age of onset varies widely among ethnic groups, ranging from 1 to 20 years (1–4).

Some authors consider direct measurement of lactase activity in the small intestinal mucosa to be the gold standard, but this technique is considered too invasive by others (5). Most clinicians prefer either the lactose breath hydrogen test (LBHT) or the lactose tolerance test for the diagnosis of lactose malabsorption. It has been reported that these tests have a low sensitivity (89%–100% LBHT, 77%–94% lactose tolerance) and specificity (69%–100% LBHT, 76%–94% lactose tolerance) (6).

Recently, in several populations, a complete correlation has been reported between the levels of lactase activity in intestinal biopsies and polymorphisms residing 13910 bp upstream of the lactase phlorizin hydrolase gene at chromosome 2q21-22. Genetic testing based on this polymorphism as a first-stage screening test for adult-type hypolactasia in all individuals older than 12 years of age (7) has been proposed. The C/C-13910 genotype shows a complete association with adult-type hypolactasia, whereas C/T-13910 and T/T-13910 genotypes are associated with lactase persistence (8). This association has been found mainly among northern Europeans, whereas in Africa hypolactasia has a more complex pattern of association and several other variants have been reported recently (9). Investigating the C/T-13910 variant and hypolactasia in Sardinia, the authors recently found an almost complete association of this variant with lactose malabsorption (10).

Genetic testing for C/T-13910 variant can be used as a diagnostic tool only when the decline of the lactase activity has been completed. Thus, knowledge of the age of onset of adult-type hypolactasia in the patient's ethnic group is essential for correct clinical use of this genetic test in the diagnosis of lactose malabsorption.

The aim of this work was to establish whether Sardinians display an age of onset of adult-type hypolactasia comparable to northern Europeans or an earlier decline of lactase activity, as observed among some Africans and other populations having a low prevalence of lactase persistence.


LBHT and genotyping of the C/T-13910 variant were performed in 392 patients (208 females, 184 males, ages 3–19 years) from the Gastroenterology Unit at the Hospital Microcitemico in Cagliari, Italy. All of the patients were from Sardinia, where adult-type hypolactasia has a prevalence of 86% (11). Of the 392 patients 9 were excluded from the study because a secondary cause of hypolactasia was found (celiac disease in 5 patients, milk allergy in 3, and Crohn disease in 1). Written informed consent to participate in the study was obtained from each adult patient or the parents of minors.


Genomic DNA was isolated from peripheral blood using a salting-out procedure (12). Genotyping of the C/T-13910 variant was performed with a polymerase chain reaction/restriction fragment length polymorphism method as previously described (13). The C/C-, C/T-, and T/T-13910 genotypes were verified by direct sequencing in 40 of 383 patients carrying the different genotypes.

Lactose and Lactulose Breath Hydrogen Test

To avoid elevated basal hydrogen production, the day before the test all of the patients consumed only rice, meat or fish, and olive oil, and were fasting for at least 8 hours before the test (14). Before and during the test, physical exercise and smoking were not permitted. Moreover, in the month preceding the LBHT, patients did not receive any antibiotic treatment. Alveolar air was collected in a gasbag, which allows separating the first 500 mL of dead space and the remaining 700 mL of end-alveolar air. To analyze breath samples, a portable handheld gastrolyzer (EC 60, Bedfont Technical Instruments, Sittingbourne, Kent, UK) was used. The accuracy of this device was ±2% of ppm, sensor sensitivity of 1 ppm, and response time under 30 seconds. The gastrolyzer's performance and reproducibility recently have been validated in comparison with a Quintron MicroLyzer (15).

Breath samples were taken at fasting and every 30 minutes for 4 hours after the administration of a 20% aqueous lactose solution (2 g/kg body weight, maximum 50 g). The test was considered positive when H2 values exceeded 20 ppm over the baseline (16).

In some subjects, to exclude low H2 excretion, a lactulose breath hydrogen test was performed with an aqueous solution containing lactulose (0.5 g/kg body weight, maximum 18 g). Breath samples were taken at fasting and hydrogen concentration analyzed every 15 minutes for 4 hours. The status of “low H2-producer” was assigned to those individuals in whom the increase of H2 did not reach 20 ppm.


The 383 patients, subdivided in 8 groups according to age, underwent lactose breath hydrogen testing and genetic testing for the C/T-13910 variant (Table 1, Fig. 1).

Frequency of the C/C-13910 genotype associated with adult-type hypolactasia and results of the LBHT in different age ranges
FIG. 1
FIG. 1:
Gray columns represent frequency of the C/C-13910 genotype; black columns represent frequency of positive LBHT for different age ranges, which are indicated on the x-axis. The line with triangles depicts the ratio between the frequency of positive LBHT and the prevalence of the C/C-13910 genotype.

The C/C-13910 genotype was found in 345 of 383 patients (frequency 0.9). As expected, no differences in the frequency of C/C-13910 genotype for each age range were found (mean 0.9, Table 1).

All 38 subjects with a not-C/C-13910 genotype had a negative LBHT, confirming normolactasia. Four of the 38 children were homozygous for the T-13910 allele (frequency 0.01), whereas the other 34 were heterozygous (frequency 0.09).

The frequency of positive LBHT results increased with patient age up to 9 years (Fig. 1). In older patients, it remained constant (frequency 0.85).


The decline of lactase activity in individuals with adult-type hypolactasia has been related to both age of onset and ethnicity (3,4). This study aimed to establish whether the age of onset of adult-type hypolactasia in Sardinians is similar to that observed in northern Europeans or if lactase activity has an earlier decline, as reported among some Africans and other populations with a low prevalence of lactase persistence (3,4). In addition, the authors wanted to establish the applicability of genetic testing for adult-type hypolactasia in Sardinian youths.

In Africans, the prevalence of adult-type hypolactasia is 70% to 90%, and the decline of lactase activity is highly variable, occurring between 1 and 8 years of age (7,17). All Thai children are lactose intolerant by 5 years of age (4). In some Mediterranean populations (eg, Greek), lactase activity declines between the ages of 5 and 12 (18). Finns' lactase activity is reduced in most children at 8 years and in all of them at 12 years (7).

Our data show that in individuals older than 9 years, the frequency of positive LBHT (85%) does not differ from that observed in healthy Sardinian adults (86%) (11). In other terms, the frequency of positive LBHT increases with age and reaches a plateau at 9 years, suggesting that in Sardinians adult-type hypolactasia becomes phenotypically expressed after this age (Fig. 1).

As expected, we found that the C/C-13910 genotype shows a frequency of about 90% in each age group. This observation indicates that the genetic test should be used on patients under 9 years of age with prudence.

In this study, the onset of decline of lactase activity as calculated by LBHT may suffer from possible errors due to a nonrandom sampling in each age range. To reduce these errors, the authors calculated the ratio between the frequency of positive LBHT and the prevalence of C/C-13910 genotype associated with adult-type hypolactasia in each age group (Fig. 1, line with triangles). The closer that this ratio is to 1, the better the phenotype corresponds to the genotype. The authors found that this ratio reached a plateau with a mean value of 0.94 only after 9 years of age, confirming the age of onset obtained by the LBHT (Fig. 1). In patients older than 9 years of age we noticed that the frequency of positive LBHT (mean 85%) was lower than that of C/C-13910 genotype (mean 90%).

The LBHT and its various modifications are indirect tests. Although a series of precautions usually are taken before performing the test, it bears a reasonably high risk for false positive and negative results (19).

In addition, because the breath hydrogen test has been reported to have a low sensitivity and because potential false positive subjects were excluded from the study, the authors supposed that a portion of the above difference could be attributed to the inclusion of false negatives (6). To resolve this discrepancy, 13 of 221 subjects older than 9 years with a C/C-13910 genotype and a negative LBHT underwent a lactulose breath hydrogen test. This test revealed that 9 individuals were not hydrogen producers. The remaining 4 patients were normal hydrogen producers.

In conclusion, we have shown that in Sardinians adult type hypolactasia is generally established in all individuals bearing the C/C-13910 genotype at 9 years of age, and that this is the correct minimum age at which to consider applying the genetic test for lactose malabsorption. The same test has to be used with prudence in patients under 9 years, in whom lactose and lactulose breath hydrogen tests remain useful in diagnosing lactose malabsorption.


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Breath hydrogen test; Genetic test; Lactose malabsorption; C/T-13910

© 2007 Lippincott Williams & Wilkins, Inc.