Low Prevalence of p.G352fsdelG Mutation in Phenylketonuria Patients from Morocco
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Low Prevalence of p.G352fsdelG Mutation in Phenylketonuria Patients from Morocco Afaf Lamzouri, 1,2 Ilham Ratbi, 1,2 Fatima Z. Laarabi, 2 Amina Barkat, 3 and Abdelaziz Sefiani 1,2 Objective: Frameshift mutation p.G352fsdelG in the PAH gene was recently reported as the most common mutation in Moroccan patients with phenylketonuria (PKU). This result, if confirmed, would considerably facilitate genetic counseling and molecular diagnosis of the disease in Morocco. Given that the incidence of PKU in the Mediterranean region is estimated at between 1/4000 and 1/10,000, this mutation would be harbored by many Moroccans. We aimed to estimate the frequency of heterozygotes for the p.G352fsdelG mutation in Moroccan newborns. Materials and Methods: In this study, we used a reliable TaqMan Ȑ real-time polymerase chain reaction to detect the mutation p.G352fsdelG in the PAH gene in 250 unrelated Moroccan newborns. DNA was extracted from umbilical cord blood with maternal consent. Results: The supposed recurrent mutation p.G352fsdelG was found in none of the 250 tested newborns. Therefore, the frequency of heterozygotes for this mutation would be less than 1/250, and the incidence of patients with PKU homozygous for this mutation would not exceed 1/100,000. Conclusion: The p.G352fsdelG mutation in the PAH gene does not appear to be prevalent in the Moroccan population and would be responsible for only few cases of PKU. The previous report of this anomaly as being responsible for 62.5% of PKU patients in Morocco could be explained by selection bias. Introduction P henylketonuria (PKU, OMIM # 261600) is a genetic disorder inherited in an autosomal recessive manner, re- sulting from mutations in the PAH gene encoding the enzyme phenylalanine hydroxylase responsible for the conversion of the amino acid phenylalanine to tyrosine (Scriver and Kauf- man, 2001). The rise of phenylalanine level in the blood allows the large passage of metabolites to the central nervous system, with a toxic effect, causing a cerebral diffuse damage (Scriver and Kaufman, 2001). The prevalence of PKU among Cauca- sians is 1/10,000 and may reach 1/4000 in other Mediterra- nean populations (Scriver and Kaufman, 2001). To our best knowledge, there are no data about the prevalence of the disease in Morocco, but considering the high level of con- sanguineous marriages (15.25%) ( Jaouad et al., 2009), it is reasonable to assume that PKU would be more prevalent in Morocco than in Caucasian populations. To date, more than 500 different disease-causing mutations have been reported in the PAH gene (PAH Locus Knowledgebase: www.pahdb.mcgill.ca). No recurrent mutation with a high frequency was reported in almost any population studied, which makes the molecular diagnosis of the disease difficult and expensive. Recently, it was reported that the most com- mon mutation in PKU patients from Morocco is the frameshift mutation p.G352fsdelG, identified in 62.5% of the studied mutant chromosomes (Dahri et al., 2010). In the same study and in a contradictory way, the authors found this mutation in a heterozygous state in only one Moroccan patient among seven living in Spain (Dahri et al., 2010). The goal of this study was to check the level of prevalence of this mutation and to better know the molecular epidemi- ology of PKU in Morocco. Materials and Methods Samples Blood samples were collected from 250 unrelated new- born’s umbilical cords. These newborn were from different regions of Morocco, with the Moroccan origin of their parents and grandparents confirmed. The study was approved by the local ethics committee, and informed consent for DNA anal- ysis was obtained from parents. Two controls with homozy- gous and heterozygous deletion p.G352fsdelG were included in this study. Methods Genomic DNA was extracted from leukocytes isolated from EDTA-anticoagulated blood using a salting-out method 1 Centre de Ge ´nomique Humaine, Faculte ´ de Me ´decine et Pharmacie, Universite ´ Mohammed V Souissi, Rabat, Morocco. 2 De ´partement de Ge ´ne ´tique Me ´dicale, Institut National d’Hygie `ne, Rabat, Morocco. 3 Centre National de Re ´fe ´rence en Ne ´onatologie et en Nutrition, Rabat, Morocco. GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 16, Number 8, 2012 ª Mary Ann Liebert, Inc. Pp. 996–998 DOI: 10.1089/gtmb.2012.0011 996
Transcript of Low Prevalence of p.G352fsdelG Mutation in Phenylketonuria Patients from Morocco
Low Prevalence of p.G352fsdelG Mutationin Phenylketonuria Patients from Morocco
Afaf Lamzouri,1,2 Ilham Ratbi,1,2 Fatima Z. Laarabi,2 Amina Barkat,3 and Abdelaziz Sefiani1,2
Objective: Frameshift mutation p.G352fsdelG in the PAH gene was recently reported as the most commonmutation in Moroccan patients with phenylketonuria (PKU). This result, if confirmed, would considerablyfacilitate genetic counseling and molecular diagnosis of the disease in Morocco. Given that the incidence of PKUin the Mediterranean region is estimated at between 1/4000 and 1/10,000, this mutation would be harbored bymany Moroccans. We aimed to estimate the frequency of heterozygotes for the p.G352fsdelG mutation inMoroccan newborns. Materials and Methods: In this study, we used a reliable TaqMan� real-time polymerasechain reaction to detect the mutation p.G352fsdelG in the PAH gene in 250 unrelated Moroccan newborns. DNAwas extracted from umbilical cord blood with maternal consent. Results: The supposed recurrent mutationp.G352fsdelG was found in none of the 250 tested newborns. Therefore, the frequency of heterozygotes for thismutation would be less than 1/250, and the incidence of patients with PKU homozygous for this mutationwould not exceed 1/100,000. Conclusion: The p.G352fsdelG mutation in the PAH gene does not appear to beprevalent in the Moroccan population and would be responsible for only few cases of PKU. The previous reportof this anomaly as being responsible for 62.5% of PKU patients in Morocco could be explained by selection bias.
Introduction
Phenylketonuria (PKU, OMIM # 261600) is a geneticdisorder inherited in an autosomal recessive manner, re-
sulting from mutations in the PAH gene encoding the enzymephenylalanine hydroxylase responsible for the conversion ofthe amino acid phenylalanine to tyrosine (Scriver and Kauf-man, 2001). The rise of phenylalanine level in the blood allowsthe large passage of metabolites to the central nervous system,with a toxic effect, causing a cerebral diffuse damage (Scriverand Kaufman, 2001). The prevalence of PKU among Cauca-sians is 1/10,000 and may reach 1/4000 in other Mediterra-nean populations (Scriver and Kaufman, 2001). To our bestknowledge, there are no data about the prevalence of thedisease in Morocco, but considering the high level of con-sanguineous marriages (15.25%) ( Jaouad et al., 2009), it isreasonable to assume that PKU would be more prevalentin Morocco than in Caucasian populations. To date, morethan 500 different disease-causing mutations have beenreported in the PAH gene (PAH Locus Knowledgebase:www.pahdb.mcgill.ca). No recurrent mutation with a highfrequency was reported in almost any population studied,which makes the molecular diagnosis of the disease difficultand expensive. Recently, it was reported that the most com-mon mutation in PKU patients from Morocco is the frameshift
mutation p.G352fsdelG, identified in 62.5% of the studiedmutant chromosomes (Dahri et al., 2010). In the same studyand in a contradictory way, the authors found this mutation ina heterozygous state in only one Moroccan patient amongseven living in Spain (Dahri et al., 2010).
The goal of this study was to check the level of prevalenceof this mutation and to better know the molecular epidemi-ology of PKU in Morocco.
Materials and Methods
Samples
Blood samples were collected from 250 unrelated new-born’s umbilical cords. These newborn were from differentregions of Morocco, with the Moroccan origin of their parentsand grandparents confirmed. The study was approved by thelocal ethics committee, and informed consent for DNA anal-ysis was obtained from parents. Two controls with homozy-gous and heterozygous deletion p.G352fsdelG were includedin this study.
Methods
Genomic DNA was extracted from leukocytes isolatedfrom EDTA-anticoagulated blood using a salting-out method
1Centre de Genomique Humaine, Faculte de Medecine et Pharmacie, Universite Mohammed V Souissi, Rabat, Morocco.2Departement de Genetique Medicale, Institut National d’Hygiene, Rabat, Morocco.3Centre National de Reference en Neonatologie et en Nutrition, Rabat, Morocco.
GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 16, Number 8, 2012ª Mary Ann Liebert, Inc.Pp. 996–998DOI: 10.1089/gtmb.2012.0011
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(Sambrook et al., 1989). DNA concentration and quality weredetermined for each sample. We searched for the mutationp.G352fsdelG on exon 10 of the PAH gene by real-time poly-merase chain reaction (PCR; Applied Biosystems 7500 FastReal-Time PCR Systems) using TaqMan� probes. This meth-od combines PCR and mutation detection in a single step. Ahybridization probe is cleaved by the 5¢ nuclease activity ofTaq DNA polymerase, only if the specific sequence is suc-cessfully amplified. Two TaqMan (PE Applied Biosystems)probes were used, one for each allele. TaqMan probes con-sisted of an 18–22-bp oligonucleotide probe that is labeledwith a reporter fluorophore at the 5¢ end and a quencherfluorophore at the 3¢ end.
Results
The p.G352fsdelG mutation in the PAH gene was not foundin any of the 250 tested newborns (Fig. 1). Carriers of thismutation in Morocco would therefore be less than one among250. According to this frequency and assuming that theMoroccan population is in the Hardy–Weinberg equilibrium,the estimated prevalence of PKU caused by the p.G352fsdelGmutation would not exceed 1/100,000.
According to Dahri et al. (2010), who report that more thanhalf of Moroccan PKU patients are carriers of thep.G352fsdelG mutation (62.5% of the mutant chromosomes),and if we consider that the prevalence of PKU in Moroccans isat least 1/10,000 (similar to Caucasians), the heterozygotefrequency for this mutation would be at least 1/70. This figurewas not confirmed by our study ( < 1/250).
Discussion
PKU is an inborn metabolic error in which metabolism ofphenylalanine into tyrosine is disrupted. An infant with PKUneeds lifelong dietary phenylalanine restriction, to ensure thedevelopment of physical and cognitive abilities. Otherwise,PKU leads to irremediable brain damage and severe mental
retardation (Gamez et al., 2007). PKU is often an overlookeddiagnosis in Morocco, and in the absence of a national new-born-screening program, the disease unfortunately leads topreventable sporadic or familial mental retardation.
Point mutations in the PAH gene are known to cause PKUin various ethnic groups, and large deletions or duplicationsaccount for up to 3% of the PAH mutations in some ethnicgroups (Desviat et al., 2006). Dahri et al. recently reported thatthe p.G352fsdelG mutation in the PAH gene is a frequentcause of PKU in Moroccans. If we assume that the prevalenceof the disease in Moroccans is at least similar to Caucasians(1/10,000), the p.G352fsdelG mutation may not be the causeof more than half of PKU in Moroccan subjects, as suggestedby the study of Dahri et al. Beside, our result is consistent withthe fact that this mutation is not significantly predominant inMoroccan patients living in France (personal communication)or among seven unrelated Moroccan patients living in Spain(the mutation p.G352fsdelG was detected in only one patient)(Dahri et al., 2010). To our best knowledge, some rare recur-rent mutations of the PAH gene have been actually reported insome populations (Guldberg et al., 1997; Chien et al., 2004;Lee et al., 2008), but not in as a large proportion of patients asthe 62.5% reported for Morocco by Dahri et al. for thep.G352fsdelG. In general, all reliable epidemiological studieshave showed a great mutational heterogeneity in PKU.Therefore, even if the study is not able to estimate the fre-quency of heterozygotes for the p.G352fsdelG mutation, be-cause of the limited size of the study population, it excludes itspresumed recurrence in Moroccans. It is possible that the se-lected patients in the previous study were from the samefamilies or regions, leading to selection bias.
Conclusion
The mutation p.G352fsdelG in the PAH gene does not ap-pear to be prevalent in the Moroccan population and wouldbe responsible for only a relatively few cases of PKU. This
FIG. 1. Curve of amplification of the study of the p.G352fsdelG mutation. (A) Normal profile; (B) heterozygous profile(control).
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result is consistent with the genetic heterogeneity at the mo-lecular level reported in most populations and highlights thatbefore announcing molecular epidemiology data, precautionsshould be taken to avoid conflicting results in the same pop-ulation. It is time for health policy makers to launch a pilotnewborn-screening program for PKU in Morocco.
Acknowledgments
The authors are indebted to Dr. Francois Feillet, Centre deReference des Maladies Hereditaires du Metabolisme, Servicede Medecine Infantile I, CHU Brabois Enfant, Allee du Mor-van 54500, Vandoeuvre, Nancy, France. Issam Khneisser isacknowledged for rereading the article.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:Ilham Ratbi, M.D.
Departement de Genetique MedicaleInstitut National d’Hygiene
27, Avenue Ibn BatoutaB.P. 769 Rabat
Morocco
E-mail: [email protected]
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