Concordance of Genetic and Breath Tests for Lactose Intolerance - Case Study Example

Critical Review of Krawczyk, M. et.al. (2008) en d Concordance of Genetic and Breath Tests for Lactose Intolerance in a Tertiary Referral Center (2008) The article is entitled Concordance of Genetic and Breath Tests for Lactose Intolerance in a Tertiary Referral Center (2008) by Marcin Krawczyk, Malgorzata Wolska, Stephanie Schwartz, Frank Gruenhage, Birgit Terjung, Piero Portincasa, Tilman Saverbaugh and Frank Lanmert. It is about Hydrogen breath test (BTH) and genotyping of a single nucleotide polymorphism (SNP) a potential methods for diagnosis of this autosomal-recessive trait. (Krawczyk, M.et.al, 2008, p. 135). This article aims to compare the result of Hydrogen Breath Test (HBT) and Genotyping of a Single Nucleotide Polymorphism (SMP) in detecting lactose non-persistence in a tertiary referral center. (Krawczyk, M.et.al, 2008, p. 136). The study concludes that the CC-genotype, BTH and genotype correlate perfectly, and the genetic test provides an unambiguous result. In both positive individual with a negative genetic test there is good reason to suspect secondary causes of lactase deficiency. (Krawczyk, M.et.al, 2008, p.138). In the study 58 consecutives patients are chosen (25 males, 33 females; median age 41 years, range 18 – 82 years) recruited prospectively between April 2005 and July 2007. (Krawczyk, M.et.al, 2008, p.137). These patients were referred by the department with non-specific gastrointestinal symptoms consistent with lactose intolerance (i.e., bloating, abdominal pain, diarrhea).All participants signed an informed consent form, and the study was conducted according to a study design approved by the local ethical committee. (Krawczyk, M.et.al, 2008, p.137). The method in genetic test includes the use of a Peripheral venous blood samples for DNA testing were obtained from all patients. DNA was isolated using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). The genotyping procedure consisted of polymerase chain reaction (PCR) amplification and SNP detection of the -13,910C>T variant using SNaPshot minisequencing (Applera, Norwalk,CT). (Krawczyk, M.et.al, 2008, p.136). For Hydrogen breathe test (BTH), it was performed after at least 12 hours overnight fasting. All patients were obliged to restrain from cigarette smoking before the test. Additionally, individuals who underwent colonoscopy or were taking any antibiotics in the fortnight before the test were excluded from the study. The test was performed after ingestion of 50 g of lactose diluted in 300 ml of water. The amount of exhaled hydrogen was measured in parts per million (ppm) before lactose ingestion (baseline), every 10 minutes during the first hour and every 20 minutes during the second and third hour of the test. In addition, the occurrence of gastrointestinal symptoms after the ingestion of lactose was monitored by questionnaires. The authors argued that taking a large amount of milk causes lactose intolerance. Consuming small amounts of milk does not exert any effects in lactose non-persistent individuals according to some authors. (Krawczyk, M.et.al, 2008, p. 135). The authors also argued that in investigating lactose intolerance it is best to measure it using the Hydrogen Breathe Test (BTH). (Krawczyk, M.et.al, 2008, p. 135). Calloway DH, et.al. (1969) and Metz,G. et.al. (1975), supports the claim of Krawczyk,M et. al. (2008), that BTH is a well-known and widely accepted procedure measures the exhaled amount of hydrogen produced in the process of fermentation once the patient is challenge with lactose. While others believed that the genetic basis for lactose intolerance, demonstrating the link between lactose persistence and a single nucleotide polymorphism (SNP) C>T located 13,910 base pairs (bp) upstream of the LCT gene in intron 13 of the minichromosome maintenance 6 (MCM6) gene. (Enattah, NS,et.al, 2002, p.233-237). Several studies claims that the genotypes TC and TT are associated with lactase persistence, whereas the CC-genotype correlates with hypolactasia.(Lember, et.al., 2006, p. 7329-7331), (Kuokkanen M.et.al., 2003,p. 647-652). Additionally, other polymorphisms (e.g. LCT –13907C>G, LCT –13915T>G) have been tested for their involvement in the onset of lactose intolerance. (Nilsson TK, Olsson LA, 2008, p.80-84). The result of the test was demonstrated by the author using table and figures. The comparison of the BTH and the genetic tests indicates 100% concordance between both procedures in CC-positive patients. (Krawczyk, M.et.al, 2008, p. 137). Concordance between BTH and genetic test shows 17 (29%) patients had positive and 41 (71%) had negative BTH results. Genotyping revealed that 15 (26%) patients were CC-positive, indicating lactose non-persistence. Accordingly, 43 (74%) patients were CC-negative, which in turn is a genetic hallmark of lactose persistence. Among these CC-negative individuals, 15 (26%) were TT homozygotes and 28 (48%) carried the heterozygous genotype TC, respectively. In Hardy-Weinberg equilibrium, all genotypes did not deviate . (Krawczyk, M.et.al, 2008, p. 137-138). The author shows the discordance between BTH and genetic test as follows: In the CC-negative group, 2 of 43 (5%) subjects tested positive in BTH, whereas the remaining 41 patients of this group showed negative results. Table II shows that sensitivity and NPV of BTH reached 100%; specificity was 95%, and PPV was 88%. These results indicate that a negative BTH is highly accurate in detecting lactase persistent subjects (carrying the T allele), whereas a positive BTH identifies all lactase non-persistent subjects (those who do not carry the T allele) and a subgroup of lactase persistent subjects in whom lactose intolerance is secondary to other causes. (Krawczyk, M.et.al, 2008, p. 137). The authors’ conclusion, is that this study demonstrates that genotyping and BTH results match perfectly in the group of lactose non-persistent individuals, yet if a patient carries a CCnegative genotype and secondary lactase deficiency is suspected, further investigations are warranted to identify the underlying diseases. (Krawczyk, M.et.al, 2008, p. 138). Similar study was done by Upton,J.et.al 2007 according to them, the identification of a simple genetic test for adult hypolactasia is a significant advance on previous methods of diagnosis. These extant methods are both time- and labour intensive, and require specialist facilities. The lactose hydrogen breath test (LHBT) is carried out in a clinical setting, and lacks both formal standardisation and sensitivity. It also requires travel to a tertiary referral centre. The authors proposes that each patient with complaints concerning lactose consumption should be investigated first by genetic tests and subsequently by additional invasive procedures to clarify if the symptoms are related to other pathologies of the gastrointestinal tract. The high prevalence of lactase nonpersistence in the general population should urge physicians to be restrictive when diagnosing primary lactose intolerance based solely on BTH results.Misdiagnosis of adult-type hypolactasia in CC-negative individuals might result in long lasting restraint from milk products. In this case it would not relieve symptoms, yet contribute to osteoporosis and other related complications. Since the costs of genetic testing do not exceed BTH, this strategy might also be cost-effective. In case of secondary lactase deficiency, accurate diagnosis and therapy will lead to reduction of lactose-related complaints and can even restore lactase activity. On the other hand, appropriate dietary recommendations are warranted for lactose intolerant individuals, since they are prone to show decreased calcium levels due to lower milk intake and impaired calcium absorption. (Terjung B,et.al. 2007, p.271- 275). In the discussion the author states the following results and analysis: The results demonstrate 100% match between BTH results and CC-positive genotypes for detection of lactose non-persistence. On the other hand, BTH and genotyping did not show perfect agreement in CC-negative individuals. Among these individuals, two subgroups can be discriminated: (i) a large number of individuals who are CC-negative and have negative BTH and (ii) a small group of CC-negative patients who suffer from secondary lactase deficiency or other conditions distorting the BTH results and who should undergo further clinical analysis. (Krawczyk, M.et.al, 2008, p. 137-138). Errors were committed during the study and were investigated using the study done by Pimental et.al. (2004). Patient should be screened first before undergoing the study failure to do so result in complications. Patient suffered from active Crohn’s disease, which can cause lactose intolerance symptoms and positive BTH results (Von Tirpitz C, Kohn C, Steinkamp M, et al,2002, 49-53). The other patient displayed several symptoms suggesting IBS. According to their study, bacterial overgrowth in the small intestine may be responsible for false positive BTH results in IBS patients (Pimental et.al. 2004, p.2700-2740). In addition, the patient was a smoker and might not have followed the recommendation not to smoke: cigarettes are known to affect BTH results . (Solomon, R. 1983, p. 29). Furthermore, heterozygous individuals (TC) express lower amounts of LCT in enterocytes compared to TT carriers . The other patient carried the genotype TC, even mild intestine abnormalities or bacterial overgrowth might have overwhelmed the existing diminished LCT activity. Still the author was able to come with a conclusion that is supported by datas they have gathered from the test and questionnaires. However, proper screening should be done first in order to avoid complications and further damage to the participant. Reference: Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L, Jarvela I. Identification of a variant associated with adult-type hypolactasia. Nat Genet 2002; 30: 233-237. Lember M, Torniainen S, Kull M, et al. Lactase non-persistence and milk consumption in Estonia. World J Gastroenterol 2006; 12: 7329-7331. Kuokkanen M, Enattah NS, 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: 647-652. Ridefelt P, Hakansson LD. Lactose intolerance: lactose tolerance test versus genotyping. Scand J Gastroenterol 2005; 40: 822-826. Nilsson TK, Olsson LA. Simulataneous genotyping of three lactose tolerance-linked polymorphisms LCT -13907C>G, LCT -13910C>T and LCT -13915T>G with Pyrosequencing technology. Clin Chem Lab Med 2008; 46: 80-84. Calloway DH, Murphy EL, Bauer D. Determination of lactose intolerance by breath analysis. Am J Dig Dis 1969; 14: 811-815. Metz G, Jenkins DJ, Peters TJ, Newman A, Blendis LM. Breath hydrogen as a diagnostic method for hypolactasia. Lancet 1975; 1: 1155-1157. Terjung B, Lammert F. Laktoseintoleranz: Neue Aspekte eines alten Problems. Dtsch Med Wochenschr 2007; 132: 271-275. Upton,J. et al, A simple gene test for lactose intolerance/adult hypolactasia Journal of the New Zealand Medical Association, 09-November-2007, Vol 120 No 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: 2700-2704. Rosenthal A, Solomons NW. Time-course of cigarette smoke contamination of clinical hydrogen breath-analysis tests. Clin Chem 1983; 29: 1980-1981. Von Tirpitz C, Kohn C, Steinkamp M, et al. Lactose intolerance in Genetic and breath tests for lactose intolerance 139 active Crohn’s disease: clinical value of duodenal lactase analysis. J Clin Gastroenterol 2002; 34: 49-53. Read More