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• Statistical points arising from Author’s Reply
  Michael E Weale
  Published on: 20 July 2005
• Author's reply
  Kaija-Leena kolho
  Published on: 4 April 2005
• DNA test for hypolactasia premature.
  D M Swallow
  Published on: 16 March 2005

• Published on: 20 July 2005

  Statistical points arising from Author’s Reply

  • Michael E Weale, Senior Lecturer

  Dear Editor,

  I write as the statistician on the paper by Mulcare and collaborators [1] which was criticised by Drs Kolho and Järvelä in their "Author's Reply" eLetter to Gut Online. I wish to correct two assertions made by Drs Kolho and Järvelä. The first is their claim that our statistical procedure “contains a risk for wrong conclusions” because “adult-type hypolactasia is difficult to assess due to inaccurate d...

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  Dear Editor,

  I write as the statistician on the paper by Mulcare and collaborators [1] which was criticised by Drs Kolho and Järvelä in their "Author's Reply" eLetter to Gut Online. I wish to correct two assertions made by Drs Kolho and Järvelä. The first is their claim that our statistical procedure “contains a risk for wrong conclusions” because “adult-type hypolactasia is difficult to assess due to inaccurate diagnostic tests”.

  This would indeed be true had we applied a ‘naïve’ test (for example, a chi-squared test) in which we had assumed the diagnoses of hypolactasia to be without error. In fact, not only did we assume that diagnoses occurred with error, but we did not even presume to know exactly what that level of error was. Instead, our uncertainty about the true level of error was modelled in a Bayesian framework and trained using available published data on comparisons of ‘true’ diagnoses (e.g. based on biopsy results) and ‘indirect’ diagnoses (e.g. based on breath hydrogen).

  To accomplish this, a novel statistical method was developed, and described in [1]. The fact that we incorporated these additional sources of error into our method means that the p-values we obtained were not as low as they would have been had we applied a naïve test such as a chi-squared test. Our remarkable finding was that, despite this, we still found significant departures in multiple sub-Saharan African populations. This led us to reject the Null Hypothesis that the presence of the C/T-13910 variant alone, even with diagnostic error, could explain the published data on lactase persistence in Africa. Other reasons must be sought to explain our results, one of which is the possibility that additional genetic variants influence lactase persistence.

  The second assertion by Drs Kolho and Järvelä was that there were “no statistics shown against the C/T-13910–variant but only speculation presented in the paper by Mulcare et al.” The meaning is unclear here. Certainly, statistics both in the sense of ‘data’ and in the sense of ‘inference’ were presented in our paper (see above). Our conclusions regarding the C/T-13910 variant were derived from carefully-constructed statistical inference, and not mere ‘speculation’.

  Reference

  1. Mulcare CA et al. The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am J Hum Genet 2004; 74:1102 -10.

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  Conflict of Interest:
  None declared.

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• Published on: 4 April 2005

  Author's reply

  • Kaija-Leena kolho, MD,PhD
  • Other Contributors:
    • Irma Järvelä

  Dear Editor,

  Dr Swallow raises a question about another DNA-variant underlying adult- type hypolactasia in sub-Saharan populations and does not recommend the analysis of the C/T-13910 variant as a genetic test in African and non-Northern European populations. Although the studies performed by us [1-3] and others [4-5] do not support the existence of another variant, we agree with Dr. Swallow that well-conducted s...

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  Dear Editor,

  Dr Swallow raises a question about another DNA-variant underlying adult- type hypolactasia in sub-Saharan populations and does not recommend the analysis of the C/T-13910 variant as a genetic test in African and non-Northern European populations. Although the studies performed by us [1-3] and others [4-5] do not support the existence of another variant, we agree with Dr. Swallow that well-conducted studies are needed to confirm this.

  The significance of the C/T-13910 variant underlying adult-type hypolactasia was questioned in the article by Mulcare and collaborators [6]. Their doubt is based on several assumptions that make it difficult to evaluate the significance of the findings. These assumptions can be listed as following:

  1) Missing phenotype data of the study subjects the presence of adult-type hypolactasia was referred to historical phenotype data that was further modified by a statistical procedure. This contains a risk for wrong conclusions. It is well documented that the clinical diagnosis of adult- type hypolactasia is difficult to assess due to inaccurate diagnostic tests and variable, usually mild symptoms [7-10]. The diagnosis is usually based on indirect tests (the lactose tolerance test or breath hydrogen test) whose specificity has been reported to range from 77% to 96% and sensitivity from 76% to 94%. Especially in children, the lactose tolerance test has not been ideal due to high rate (up to 30%) of false positive results [11]. The specificity of the breath hydrogen test varies between 89-100% and sensitivity 69-100% [12]. There is evidence that the breath hydrogen test may be an indicator of bacterial overgrowth rather than lactose malabsorption [13].

  2) Definition of the ethnic origin was based on self-definition and spoken language. As the authors themselves clarify, African populations have complex demographic histories. Many of the analysed groups were very small making the chance to play a role. In contrast to the findings by Mulcare et al. our genotyping data in nomadic pastoralists Fulani-Sudanese is in agreement with the previously published figures of lactase persistence in this population.

  3) There is no statistics shown against the C/T-13910 –variant but only speculation presented in the paper by Mulcare et al. [6].

  When conducting phenotype-genotype correlation studies in lactase persistence/non persistence detailed clinical studies are essential. The studies are difficult since it is unethical to take an intestinal biopsy from healthy subjects that would give the most reliable diagnosis. Measurement of lactase activity from hospitalised patients with a clinical indication for intestinal biopsy may reflect a disease in the gut and the result obtained might not correlate with the genotype. These uncertainties should be taken into account when interpreting the genotyping results in adult-type hypolactasia.

  References

  1. Enattah NS et al. Identification of a variant associated with adult -type hypolactasia. Nat Genet 2002;30:233-37.

  2. Kuokkanen M, Enattah N, Oksanen A, Savilahti E, Orpana A, Jarvela I. Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia. Gut 2003; 52:647 -52.

  3. Rasinpera H et al. A genetic test which can be used to diagnose adult- type hypolactasia in children. Gut 2004; 53:1571-76.

  4. Olds LC and Sibley E (2003) Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element. Hum Mol Genet 2003;12:2333-40.

  5. Troelsen JT et al. An upstream polymorphism associated with lactase persistence has increased enhancer activity. Gastroenterology 2003; 125: 1686-94.

  6.Mulcare CA et al.The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am J Hum Genet 2004;74:1102 -10.

  7. Carroccio A, Montalto G, Cavera G, Notarbatolo A. Lactose intolerance and self-reported milk intolerance: relationship with lactose maldigestion and nutrient intake. J Am Coll Nutr 1998;17:631-36.

  8. de Vrese M, Stegelmann A, Richter B, de Vrese M, Stegelmann A, Richter B et al. Probiotics – compensation for lactase insufficiency. Am J Clin Nutr 2001; 2:421-29.

  9. Saltzman JR, Russell RM, Golner B, Barakat S, Dallal GE, Goldin BR. A randomized trial of Lactobacillus acidophilus BG2FO4 to treat lactose intolerance. Am J Clin Nutr 1999;69:140-46.

  10. Bohmer CJ Tuynman, HA. The effect of a lactose-restricted diet in patients with a positive lactose tolerance test, earlier diagnosed as irritable bowel syndrome: a 5-year follow-up study. Eur J Gastroenterol Hepatol 2001; 13:941-44.

  11. Krasilnikoff PA, Gudmand-Hoyer E, Moltke HH. Diagnostic value of disaccharide tolerance tests in children. Acta Paediatr Scand 1975;64:693- 98.

  12. Arola H. Diagnosis of hypolactasia and lactose malabsorption. Scand J Gastroenterol Suppl. 1994;202:26-35.

  13. Pimentel M, Kong Y, Park S. Breath testing to evaluate lactose intolerance in irritable bowel syndrome correlates with lactulose testing and may not reflect true lactose malabsorption. Am J Gastroenterol 2003;98:2700-4.

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  Conflict of Interest:
  None declared.

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• Published on: 16 March 2005

  DNA test for hypolactasia premature.

  • D M Swallow, Professor

  Dear Editor,

  I write in response to the recently published article by Rasinperä and colleagues [1] in which a DNA test is proposed for ‘adult type hypolactasia’.

  The ability to digest the milk sugar lactose as an adult (lactase persistence) is a variable genetic trait in human populations, lactase persistence being the most frequent phenotype in northern Europe, while lactase non-persistence or ‘adult...

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  Dear Editor,

  I write in response to the recently published article by Rasinperä and colleagues [1] in which a DNA test is proposed for ‘adult type hypolactasia’.

  The ability to digest the milk sugar lactose as an adult (lactase persistence) is a variable genetic trait in human populations, lactase persistence being the most frequent phenotype in northern Europe, while lactase non-persistence or ‘adult type hypolactasia’ is more frequent in most other populations [2]. In sub-Saharan Africa for example, lactase persistence is found only at low frequency in the majority of populations that have been tested, but in some populations, particularly pastoralist groups, it is significantly more frequent.

  A CT polymorphism located 13.9kb upstream of exon 1 of the lactase gene (LCT) was previously shown in a Finnish population to be tightly associated with the lactase persistence phenotype [3] and it is this change that is proposed as a DNA test for both Europeans and Africans. We agree that the presence of a T at this polymorphic site is indeed a fairly good predictor of lactase persistence in northern Europeans [4], and there is evidence that this nucleotide resides in a functional element [5,6]. However the presence of the alternative allele C at this site is not a good predictor of lactase non-persistence or ‘adult hypolactasia’ in many non northern Europeans [7,8].

  I particularly draw readers attention to our recent study [8]. We typed this polymorphism in 1671 individuals from seven African countries, which included 20 distinct cultural groups. In seven cases it was possible to match the groups tested with groups from the literature for whom phenotypic information is available. In five of these groups the published frequencies of lactase persistence are >25%. We found the T allele in Cameroon but it was so rare elsewhere that it cannot explain the frequency of the lactase persistence phenotype throughout Africa and we devised a statistical test to show that these results were unlikely to have been obtained by chance.

  Our ongoing results support this published information and we urge the community to refrain from using DNA tests on Africans and probably other non northern Europeans until an appropriate DNA change has been identified.

  References

  1. Rasinpera H et al. (2004) A genetic test which can be used to diagnose adult-type hypolactasia in children. Gut 53 (11):1571-1576.

  2. Swallow DM (2003) Genetics of lactase persistence and lactose intolerance. Annu.Rev.Genet. 37:197-219.

  3. Enattah NS et al. (2002) Identification of a variant associated with adult-type hypolactasia. Nat.Genet. 30 (2):233-237.

  4. Poulter M et al. (2003) The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans. Ann.Hum.Genet. 67 (Pt 4):298-311.

  5. Olds LC and Sibley E (2003) Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element. Hum.Mol.Genet. 12 (18):2333-2340.

  6. Troelsen JT et al. (2003) An upstream polymorphism associated with lactase persistence has increased enhancer activity. Gastroenterology 125 (6):1686-1694.

  7. Bersaglieri T et al. (2004) Genetic signatures of strong recent positive selection at the lactase gene. Am.J.Hum.Genet. 74 (6):1111-1120.

  8. Mulcare CA et al. (2004) The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene (LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am.J.Hum.Genet. 74 (6):1102-1110.

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  Conflict of Interest:
  None declared.

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