Phenylketonuria in Spanish Gypsies: Prevalence of the IVS10nt546 mutation on haplotype 34

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  • 66 DESVIAT ET AL. HUMAN MUTATION 9:6668 (1997)

    ' 1997 WILEY-LISS, INC.

    HUMU 000


    Phenylketonuria in Spanish Gypsies: Prevalence ofthe IVS10nt546 Mutation on Haplotype 34Lourdes R. Desviat, Beln Prez, and Magdalena Ugarte*Centro de Biologa Molecular Severo Ochoa, CSIC-UAM, Universidad Autnoma de Madrid, Madrid, Spain;Fax: 34-1-734-7797

    Communicated by Jrgen Horst

    on a different RFLP and STR haplotype backgroundthan in Spanish non-Gypsies.


    In this study we have examined eight Gypsy PKUfamilies from different communities in Spain. Two ofthe families are related. Consanguinity among par-ents has not been proved. Overall, 11 affected indi-viduals, among them three pairs of siblings, werestudied. One of the patients, now 3 months old, had13 mg Phe/dl at diagnosis; the rest of the patientshad Phe values of >30 mg/dl. In most cases, patientsdo not follow treatment adequately, and the clinicaloutcome varies. In two treated patients, Phe toler-ance was 350370 mg/day.

    A total of 23 non-Gypsy unrelated PKU patientsfrom different regions of Spain were also included inthe study of the STR alleles. All carry mutationIVS10nt546: 8 in homozygous fashion and 15 in het-erozygous fashion. The phenotypic classification andthe haplotype analysis of the patients were previouslydescribed (Martnez-Pardo et al., 1994; Desviat etal., 1993).

    PCR amplification was performed using driedblood spots as the source of DNA, as described pre-viously (Prez et al., 1993). The IVSl0nt546 muta-tion was detected by digestion of amplified exon 11with DdeI. The R252W mutation was detected bydigestion of amplified exon 7 with AvaI.

    RFLP in the PAH gene were determined by South-ern blotting and hybridization with a PAH cDNAprobe (for the EcoRI and EcoRV polymorphisms),PCR and restriction enzyme digestion (for BglII,PvuII(a), PvuII(b), MspI, and XmnI polymorphisms),and PCR and electrophoretic examination of theVNTR alleles in the 3 end of the PAH gene (Goltsov

    Received 6 April 1995; accepted 7 July 1995.

    *Correspondence to Magdalena Ugarte.


    Phenylketonuria (PKU) is an autosomal recessivedisorder caused by a deficiency of the liver enzymephenylalanine hydroxylase (PAH) gene, which, leftuntreated, causes mental retardation. The PAH genehas been cloned and sequenced (Kwok et al., 1985);to date, more than 200 different mutations have beendetected (PAH Mutation Analysis Consortium). Indifferent human populations, associations betweenmutations and restriction fragment length polymor-phism (RFLP) haplotypes, defined by seven diallelicpolymorphisms and a VNTR system in the PAH gene(Eisensmith and Woo, 1992) have been described.Recently, an STR polymorphism was identified inintron 3 of the PAH gene (Goltsov et al., 1993), add-ing versatility in the study of the genetic heteroge-neity of PKU.

    Gypsies are believed to have originated in India.They moved to Europe about 500 years ago, wherethey still live in nomadic or sedentary communities,maintaining their own way of life apart from the hostpopulations. They represent a high-risk populationfor genetic diseases such as PKU, due to their highdegree of consanguinity. Recently, a mutationalanalysis in Gypsy PKU families from Slovakia was per-formed (Kalanin et al., 1994), and mutation R252Wwas found in all the mutant alleles examined.

    Spanish Gypsies present a unique HLA antigendistribution and are genetically closer to Indian popu-lations than to the Spanish non-Gypsy population(de Pablo et al., 1992). They also show a consider-able similarity to Hungarian and Czech Gypsies (dePablo et al., 1992). In this study, we have investi-gated the genetic origin of PKU in the Spanish Gypsypopulation, analyzing mutations and polymorphismsin the PAH gene. The major Mediterranean muta-tion. IVS10nt546 (Dworniczak et al., 1991), wasfound in 87.5% of the Gypsy mutant alleles studied,


    et al., 1992). In many cases, the availability of onlydried blood spots from the proband and family pre-cluded the examination of the EcoRV and EcoRI poly-morphisms. The haplotype was inferred, taking intoaccount the polymorphisms examined and the mu-tationhaplotype associations observed in the com-pletely haplotyped patients.

    The alleles in the STR system in the 3 end of thePAH gene (Goltsov et al., 1993) were amplified witha fluorescent primer and analyzed using A.L.F. DNASequencer and Fragment Manager (Pharmacia).Fluorescent internal-lane standards were used as sizemarkers. The allele length reflects the true size ofthe fragments and thus is 2 bp shorter than the allelelengths given by Goltsov et al. (1993), as describedby Zschocke et al. (1994).


    A total of 11 Gypsy PKU patients were screenedfor the presence of IVS10nt546. Among them, 8 werefound to be homozygous for this mutation and 3 het-erozygous. Mutation R252W was found in two het-erozygous siblings. The results were confirmed in theparents. In the remaining heterozygous patient, theother mutant allele remains unidentified.

    All the homozygous patients for IVS10nt546 andthe compound heterozygotes IVS10nt546/ R252Wexhibit a severe PKU phenotype: Phe at diagnosis>30 mg/dl. The patient heterozygous for IVSl0nt546and an unidentified mutation has a milder pheno-type, with a Phe at diagnosis of 13 mg/dl.

    The analysis of polymorphisms in the PAH geneshowed that IVSl0nt546 in Spanish Gypsies is asso-ciated with haplotype 34. 7. In patients in whom com-plete haplotyping was not possible, the 7 polymorphicsites examined coincide with haplotype 34.7. All theGypsy chromosomes with IVS10nt546 were foundon the background of the 230-bp STR allele. Theresults are summarized in Table 1.

    In Spanish non-Gypsies, mutation IVSl0nt546 isassociated with haplotype 6.7. Examination of theSTR alleles showed that most of the chromosomes

    (17/31) carry the 250-bp allele. The rest of theIVS10nt546 chromosomes are distributed among theSTR alleles of 248 bp (6/31), 256 bp (5/31), 260 bp(2/31), and 264 bp (1/31). We have not found anychromosome with the 230-bp STR allele.


    Gypsies are a genetically isolated group historicallyconsidered to be of Indian origin. Both PKU muta-tions described in Gypsies up to now, IVSl0nt546 andR252W (frequent in the Mediterranean), have prob-ably been transferred to Gypsies from the caucasianpopulation, as Mediterranean countries were the firstamong which Gypsies passed through or settled in.

    In the sample studied from Spanish Gypsies, themajor mutation causing PKU was found to beIVS10nt546, associated with haplotype 34, theVNTR allele of 7 repeats and the STR allele of 230bp. Although prevalently associated with haplotype6 in all the populations studied, mutation IVS10nt546has also been detected on other haplotypes, amongthem haplotype 34 (Tyfield et al., 1993), which isidentical to haplotype 6, except for the PvuII(b) poly-morphic site. This haplotype could have originatedfrom haplotype 6 by a point mutation. In Gypsies,the STR allele in IVS10nt546 chromosomes is alsodifferent and unique when compared with Spanishnon-Gypsies. This could be explained by a deletionmutation in the STR system. Tetranucleotide repeatshave a high average mutation rate, although muta-tions involving loss of more than a pair of repeat unitsare unfrequent (Weber and Wong, 1993). Alterna-tively, a single recombination event after the PvuII(b)site, between a mutant haplotype 6 and a normalhaplotype 34 with the 230-STR bp allele, could ex-plain the origin of the Gypsy alleles with IVS10nt546.

    The data obtained suggest a single origin for allthe Spanish Gypsy IVS10nt546 alleles, which spreadamong the Gypsy population favored by their geneticisolation and close inbreeding. The different resultsobtained in the study with Gypsies from Slovakia(Kalanin et al., 1994) could be explained by the low

    TABLE 1. Analysis of Polymorphisms in the PAH Gene in Gypsy Chromosomes from Spain With IVS10nt546a

    PAH polymorphismsHindIII

    No. of alleles Mutation BglII PvuIIa STR PvuIIb EcoRI MspI XmnI VNTR EcoRV Haplotype

    2 IVS10nt546 + 230 + + 7 34.712 IVS10nt546 + 230 ND + 7 ND 34.7b

    1 R252W + 242 ND + 3 ND ND1 ND 242 ND + 8 ND ND

    ND, not determinedaSiblings with identical genotype were considered as one patient.bMost plausible haplotype, taking into account the polymorphisms examined and the mutation haplotype association observed.


    number of alleles examined, both in Slovakia and inSpain. Examination of a larger sample could revealsimilarities between both populations. In fact, muta-tion R252W was also detected in one Spanish Gypsyallele, and both mutation IVS10nt546 on haplotype6 and mutation R252W have been described in Gyp-sies from Bulgaria (Kalaydjieva et al., 1993). Analy-sis of other Gypsy communities in Mediterraneancountries will provide further information on the ori-gin of the Spanish Gypsy IVS10nt546 allele.

    The simple diagnostic test employed to detectIVS10nt546 may aid in the correct diagnosis ofthe disease, as well as enabling carrier detectionand direct prenatal diagnosis in the Spanish Gypsypopulation.


    The authors thank M.J. Garca for collaboratingwith the phenotyping and the following pediatricians/investigators for sending samples: Dr. Martnez-Pardo(Madrid), Dr. Vilaseca (Barcelona), and Dr. Cardesa(Badajoz). The financial support of the FundacinRamn Areces to the Centro de Biologa MolecularSevero Ochoa is gratefully acknowledged. This workwas supported by grant SAF-93-0076 from theCICYT (Comisin Interministerial de Ciencia yTecnologa).


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