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Perspectives for Early Genetic Screening of Lactose Intolerance: - 13910C/T Polymorphism Tracking in the MCM6 Gene

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Perspectives for Early Genetic Screening of Lactose Intolerance: - 13910C/T Polymorphism Tracking in the MCM6 Gene 66 The Open Biology Journal, 2010, 3, 66-71 Open Access Perspectives for Early Genetic Screening of Lactose Intolerance: - 13910C/T Polymorphism Tracking in the MCM6 Gene 1 2 *,1 1 Marta A.S. Arroyo , Ana Cláudia P. Lopes , Vania B. Piatto and José Victor Maniglia 1Department of Otorhinolaryngology of Medical School of São José do Rio Preto (FAMERP), Brazil 2Department of Morphology of Medical School of São José do Rio Preto (FAMERP), Brazil Abstract: Introduction: For many years Lactose intolerance has been, considered as a universal problem in many children and adults. Objective: The aim is to investigate the prevalence of polymorphism -13910C/T, in a neonatal tracking, for early diagnosis of lactose tolerance/intolerance. Materials and Methods: In a cross-sectional study of 310 Brazilian newborns, DNA was extracted from leukocyte umbilical cord and specific primers were used to amplify the region that encloses the -13910C/T polymorphism of the MCM6 gene, using the polymerase chain reaction and the restriction fragment length polymorphism tests. Results: One hundred and sixty (52%) male newborns and 150 (48%) female new borns were evaluated. Out of these, 191 (62%) presented CC genotype (lactose intolerant), 95 (31%) CT genotype, and 24 (7%) TT genotype, comprising a total of 119 (38%) lactose tolerant newborns. Accordingly the newborns´ gender distribution in relation to the phenotypes has been found; 97 (32%) of male gender and 94 (30%) of female gender lactose intolerant, and 63 (20%) male and 56 (18%) female lactose tolerant newborns, not being such distribution statistically significant (p = 0.801). Conclusions: The molecular analysis made possible the identification of the presence or absence of lactase persistence variant in the Brazilian newborns. The neonatal molecular diagnosis can optimize the follow-up of positive results in newborn screening for lactose intolerance. Keywords: Molecular analysis, MCM6 gene, lactase, lactose malabsorption, hypolactasy. INTRODUCTION below five years of age have the evidence of lack of lactase and lactose malabsorption while white children, typically, do Approximately 70% of the world population has lactase not develop lactose intolerance symptoms up to the age of primary deficiency. This percentage varies according to four to five years [1-4]. ethnicity and is related to the use of dairy products in the diet, resulting in the genetic selection of individuals with the Despite many studies have been carried out worldwide ability to digest lactose. In populations with the predomi- [1,3-7], there are few data published pertaining the lactose nance of dairy products in their diet due to the pastoral intolerance prevalence in the Brazilian population. Accor- tradition, particularly, in the North of Europe, only 2% of the ding to these data, there has been a frequency of approxi- population has primary lactase deficiency. Contrarily, accor- mately 50% in the Caucasian, 85% in the Negroids, and ding to the anthropologic studies, people with high preva- 100% in the Orientals, being these results, in accordance lence of adult lactose malabsorption are those, who have with the ethnic race of the Brazilians and according to the agricultural and hunting tradition and who never drank milk studied region [8-12]. or who started taking it only a few thousand year ago, but as Adult lactose malabsorption is hereditarily inherited by fermented dairy products and, therefore, poor in lactose [1- autosomal recessive gene. The opposite condition - the per- 4]. sistence of lactase activity in adult life - is inherited by Primary deficiency prevalence is from 50% to 80% in the autosomal dominant gene and it is probably a polymorphism Hispanic population, 60% to 80% in Negroids, mainly in a regulatory gene, which results in permanent capacity of Africans from Hamite origin, and Ashkenazi Jewish people digesting sugar from milk all lifelong. The persistence of and almost 100% in the Americans and Asian Indians, lactase activity represents a genetic polymorphism in the besides the Eskimos. The groups with intermediate preva- human population that involves the regulation of deve- lence are cross-breed from the groups mentioned: African lopment [13,14]. cross-breed between Bantu and Hamite, cross-breed from the Several polymorphisms have been found in introns and Europeans with Orientals and with Indians. The beginning exons of the lactase gene and in its promoter region, but age and the prevalence differ among many populations. none of them, consistently correlates with the persistence/ Approximately 20% of Hispanic, Asians and Black children non-persistence of lactase [15]. A recent finding has been described by a group from Finland, in which two polymor- phisms have been found in the introns 9 and 13 of the *Address correspondence to this author at the Department of Otorhinolaryngology of Medical School of São José do Rio Preto, minichromosome maintenance 6 gene (MCM6, OMIM – (FAMERP), São Paulo, Brazil, Av. Brigadeiro Faria Lima, nº 5416, Vila 601806) [13]. The polymorphisms, numerated from the São Pedro, São José do Rio Preto, SP, Zip Code: 15090-000, Brazil; beginning codon – ATG – from LCT gene, are the -13910C/ Tel: +55-17-32015747; E-mail: [email protected] T in intron 13, located 14 Kb upstream of the lactase gene 1874-1967/10 2010 Bentham Open Perspectives for Early Genetic Screening of Lactose Intolerance The Open Biology Journal, 2010, Volume 3 67 and the polymorphism -22018G/A in intron 9, located 22 Kb (F) - AAG ACG TAA GTT ACC ATT TAA TAC (26533 to upstream of the gene, which correlate 100% and 97% with 26556 position) and reverse primer MCM6-LCT (R) - CGT the lactose tolerance/intolerance phenotype [13, 16, 17] res- TAA TAC CCA CTG ACC TAT CCT (26748 to 26725 pectively. The homozygote genotypes CC and GG have low position), and the conditions for the PCR reaction were lactase levels (non-persistent to lactase) therefore being according to those described in the literature [21], with intolerant to lactose. The homozygote genotypes TT and AA changes in the annealing temperature, in the cycles number have high levels of lactase (lactase persistent) and the hetero- of PCR and in the duration of cycles. Genomic DNA (200 zygotes CT and GA intermediate lactase levels, being all of ng) was used as template in a reaction of final volume of 25 them tolerant to lactose. Therefore, the T allele is present in µL, containing 10 pmol of each primer and all other reagents all individuals with lactase persistence (tolerant to lactose) from FideliTaqTM PCR MasterMix (2X) (GE Healthcare®), and absent in those with non-persistence (lactose intolerant) according to manufacturer’s protocol. The amplification was [18]. performed as follows: initial denaturation at 94ºC for 3 min followed by 35 cycles of denaturation at 94ºC for 1 min, The MCM6 gene has been scanned by fluorescence in annealing at 60ºC for 1 min and extension at 72ºC for 2 min. situ hybridization in chromosome 2, in position 2q21 [19], The final extension at 72ºC was for 10 min. being expressed in a variety of adult and fetal human tissues, having an important role in the regulation of the DNA As a product for the PCR reaction, a fragment of 216 bp replication. The lactase persistence/non-persistence is, there- was amplified, which was, afterwards, submitted to the fore, associated with a non-coding variation in the MCM6 restriction analysis by the RFLP test, using the BsmFI [21] gene situated upstream of the lactase gene, in a region that enzyme (New England Biolabs)®, at 65°C, for 2h30min. appears to act as a cis element capable of enhancing diffe- When the polymorphism is present in both alleles (homo- rential transcriptional activation of the lactase promoter zygote sample), the PCR product is digested by the BsmFI region [14]. enzyme, due to the recognition of the enzymatic restriction site, in fragments of 126 bp and 90 bp and, when the poly- Although DNA analyses, specific in allowing T/C and morphism is present in only one allele (heterozygote G/A polymorphisms identification, have made possible the sample), the product of the PCR of the mutant allele is elucidation of the molecular base involved in the regulation digested in two fragments of 126 bp and 90 bp and of the of lactase promoting transcription (LCT), with consequent normal allele is not digested, presenting a fragment of 216 importance in the diagnosis of persistence/non-persistence of lactase activity, there are few studies in developing coun- bp. In the polymorphism absence, in both alleles, the PCR product is not digested, presenting only the 216 bp fragment, tries, especially in Brazil [20-22] and, up to now, none on because there is no recognition of the enzyme restriction site, newborns. due to the non substitution of the nitrogenate bases C/T, in This study aimed at investigating the prevalence of - the position -13910 of the MCM6 gene. 13910C/T polymorphism in a neonatal screening, for an early diagnose of lactase tolerance/intolerance in order to The products from both reactions, PCR and RFLP, were analyzed by agarose gel electrophoresis 2% in TBE 1x provide adequate specific therapeutic measures. buffer, containing ethidium bromide, in 0.5µg/mL concen- tration, submitted to ultraviolet lighting, to assure the success MATERIALS AND METHODS of the reaction and the gel was photodocumented. During the period from July to October 2009 a cross- Statistical Analysis sectional study was carried out, in which, 310 both male and female newborns were evaluated by molecular tests,
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