Hum Genet (1992) 89:69-72

�9 Springer-Verlag 1992

Linkage disequilibrium between phenylketonuria and RFLP haplotype 1 at the phenylalanine hydroxylase locus in Portugal

Catherine Caiilaud 1, Laura Vilarinho 2, Antoine Vilarinho 2, Fran~oise Rey 1, Monique Berthelon 1, Rosario Santos 2, Stanislas Lyonnet 1, Marie-Louise Briard 1, Rui Vaz Osorio 2, Jean Rey 1, and Arnold Munnich 1

1Unit6 de Recherches sur les Handicaps G6n6tiques de l'Enfant INSERM U-12, H6pital des Enfants-Malades, 149, Rue de S~vres, F-75743 Paris Cedex 15, France 2Instituto de Genetica Medica Jacinto de Magalhaes, Praga Pedro Nunes 74, P-4000, Porto, Portugal

Received June 5, 1991

Summary. RFLPs of 36 normal and 41 mutant alleles at the phenylalanine hydroxylase locus were determined in 31 Portuguese kindreds. A total of 14 haplotypes includ- ing 10 normal and 7 mutant alleles were observed. Al- most 75% of all mutant alleles were confined within only two haplotypes, namely haplotype 9 (17.1%) and hap- lotype 1 (56.1%). This frequency of mutant haplotype 1 in Portugal is, to our knowledge, the highest for this mu- tant haplotype in all studies reported to date. Other mu- tant haplotypes were either rare (haplotype 2, 9.7%) or totally absent (haplotype 3, 0%). Only 24.5% of all mu- tant alleles were found to consistently carry identified mutations, particularly R261Q (9.8%), R252W (3.3%), R408W (1.6%) and AI94 (3.3%). A new mutation, L249F, located in the seventh exon of the gene, accounted for 6.5% of all mutant alleles in our series. Interestingly, this mutant genotype was consistently associated with mutant haplotype 1 ( P < 0 . 0 1 ) , as also observed for the R261Q mutation. It appears, therefore, that mutant hap- lotype 1 is genotypically heterogeneous in Portugal and that more than two mutations account for its prevalence in this country.

Introduction

Classic phenylketonuria (PKU) and variant forms of hy- perphenylalaninemias are caused by a deficiency of the liver-specific enzyme phenylalanine hydroxylase (PAH; E.C.1.14.16.1). The isolation of the gene and the pro- duction of a full-length cDNA for P A H (Kwok et al. 1985) has facilitated the description of a number of dif- ferent mutations and the characterization of many re- striction fragment length polymorphism (RFLP) haplo- types at the P A H locus (Chakraborty et al. 1987). Initial data showed that specific mutant genotypes are strongly linked to particular RFLP haplotypes (DiLella et al. 1986; 1987). However , when more PAH-deficient alleles were studied, it became increasingly apparent that a par-

Offprint requests to: C. Caillaud

ticular RFLP haplotype may carry different mutations and, conversely, that a given mutation may be associated with different haplotypes at the P A H locus (Berthelon et al. 1991) in Mediterranean countries.

In order to further investigate the genetic heteroge- neity of P A H deficiencies in southwestern Europe, a group of 31 PKU children from Portugal was screened for eight different mutations by polymerase chain reac- tion (PCR) amplification of their genomic D N A and hy- bridization with allele-specific oligonucleotides. More- over, in order to study the genotype-haplotype associa- tions, RFLP haplotypes at the P A H locus were deter- mined for 41 PKU chromosomes.

Surprisingly, one mutant haplotype, haplotype 1, was prevalent, but genotypically heterogeneous, in Portugal.

Materials and methods

Patients

A series of 31 unrelated families, each with at least one child af- fected with permanent hyperphenylalaninemia, was investigated (plasma phenylalanine levels over 15mg/100ml on neonatal screening, dietary phenylalanine tolerance below 450 mg/day). Pa- tients were evaluated either in the Instituto de Genetica Medica, Porto, Portugal or in the Department of Pediatrics, H6pital des Enfants-Malades, Paris, France. The gene pool studied comprised 61 mutant alleles. Of all mutant genes, 59 originated in northern Portugal (Porto area) and 2 in the Lisbon area. Two families were of mixed French-Portuguese ancestry and two families were con- sanguineous.

Methods

For RFLP analysis, genomic DNA isolated from blood leukocytes was digested using seven restriction enzymes (PvuII, BglII, XmnI, MspI, HindIII, EcoRV and EcoRI+BamHI) and analysed by Southern blotting. The full-length PAH cDNA was used as a probe for hybridization (Kwok et al. 1985) and complete RFLP haplotypes were established for each family member and classified (Woo 1988). Unambiguous determination of haplotypes was possi- ble in 21 of 31 families for a total of 77 chromosomes (36 normal alleles and 41 mutant alleles). Comparison of RFLP and haplotype

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Table 1. Haplotype frequencies at the PAH locus in Portugal. NS, Not significant

Haplo- Normal alleles Mutant alleles Statistical types Number % Number % comparison

1 11 30.5 23 56.1 P<0.05 2 2 5.5 4 9.7 NS 3 1 2.8 0 0.0 NS 4 9 25.0 1 2.4 P < 0.01 5 5 13.9 0 0,0 P < 0,05 6 0 0.0 2 4.9 NS 7 4 11.1 0 0.0 NS 9 0 0.0 7 17.1 P < 0.05

12 1 2.8 0 0.0 NS 16 1 2.8 0 0.0 NS 34 0 0.0 2 4.9 NS 39 0 0.0 2 4.9 NS A 1 2.8 0 0.0 NS B 1 2.8 0 0.0 NS

Total 36 41

frequencies between normal and mutant alleles was made using the chi-square test with Yate's correction when necessary.

For allele-specific oligonucleotide (ASO) screening of muta~ tion-containing regions, amplification primers, ASO probes and experimental procedures were as previously described, namely: (1) IVS-12 (DiLella et al. 1986), (2) AI94 (Caillaud et al. 1991), (3) R261Q (Abadie et al. 1989), (4) R252W (Abadie et al. 1989), (5) E280K (Lyonnet et al. 1989). Screening for the L311P muta- tion was performed by Southern blotting via detection of the spe- cific MspI pattern (Lichter-Konecki 1988a).

For characterization of the L249F mutation, amplified exon 7 was cloned in phage M13 mpl8 and sequenced by the dideoxy chain-termination method. Subsequent screening for this mutation was performed using the following ASO probes: normal probe, 5' GCTGGCCTGCTTTCCTCTC 3'; mutant probe, 5' GAGAGG- AAAACAGGCCAGC 3'. Temperatures for hybridization and washing were 58~ and 64~ respectively.

R e s u l t s

R F L P haplotypes and fi'equencies in normal and mutant P A H genes in Portugal

A to ta l of ten R F L P hap lo types was o b s e r v e d a m o n g n o r m a l P A H genes (Tab le 1). Two of these hap lo types have not been r e p r e s e n t e d in ped ig ree s r e p o r t e d to da te ( the + symbol refers to the l ightest a l le le) :

A + + - + + - + + B + + - - + - - +

A l t h o u g h ten d i f ferent hap lo types were o b s e r v e d for the no rma l genes , a lmost 70% of no rma l a l le les were con- f ined to only th ree of t hem (hap lo types 1, 4 and 5). A m o n g the mu tan t al leles, only seven d i f ferent hap lo-

Table 2. Distribution of PKU mutations in 61 mutant PAH genes. ND, Not determined; NH, complete haplotype impossible to de- termine

Mutation Alleles Haplotypes

Num- % ber

1 2 34 NH N D

R261Q 6 9.8 R252W 2 3.3 R408W 1 1.6 AI94 2 3.3 L249F 4 6.5 E280K 0 0.0 L311P 0 0.0 IVSI2 0 0.0

4 1 1 1 1

i

Total 61 24.5

types were obse rved . In te res t ing ly , more than 70% of m u t a n t P A H genes were conf ined within only two R F L P hap lo types , namely I and 9. H a p l o t y p e 1 alone accounted for 56% of m u t a n t P A H genes ins tead of the 30.5% found in n o r m a l P A H genes ( P < 0.05). A s t rong associ- a t ion b e t w e e n h y p e r p h e n y l a l a n i n e m i a s and pa r t i cu la r R F L P s was cons is ten t ly a p p a r e n t at the popu la t i on level for res t r ic t ion enzymes Xm n I ( P < 0 . 0 0 1 ) , MspI ( P < 0.001), HindIII (P < 0.02) and Eco RV (P < 0.05). It is wor th not ing tha t the same hap lo type accounts for abou t 31% of all m u t a n t al le les in F rance (P < 0.05, R e y et al. 1988).

PCR screening for PKU mutations m Portugal

T a b l e 2 shows that only one qua r t e r (24 .5%) of all P K U al leles t es ted were found to car ry an iden t i f i ed mu ta t i on in our series. One m u t a t i o n was more f requen t ly rep- r e sen ted , name ly R261Q (9 .8%, A b a d i e et al. 1989; O k a n o et al. 1990). The AI94 muta t ion , or ig inal ly de- scr ibed in Por tuga l , was only found twice in our s tudy (Cai l laud et al. 1991). In te res t ing ly , muta t ions at the splice d o n o r site of in t ron 12 (IVS12) and at codon 408 (R408W) , which account for 60% of all P K U al leles in no r the rn E u r o p e ( D i L e l l a et al. 1986, 1987), were al- most comple te ly absent in Por tuga l (IVS12, 0% ; R408W, 1 .6%). Similar ly , the E280K m u t a t i o n that accounts for 6.3% of m u t a n t al le les in wes te rn E u r o p e and Nor th Af - rica (Be r the lon et al. 1991) was not found in the p resen t s tudy.

Identification of a new mutation in PKU (L249F) and its association with mutant haplotype 1

In an a t t e mp t to charac te r i ze the geno type ( s ) assoc ia ted with m u t a n t h a p l o t y p e 1 at the P A H locus in Por tuga l , the ampl i f i ed exon 7 of th ree af fec ted ch i ldren was sub- c loned and sequenced . A C- to -T t rans i t ion at codon 249 changed leucine (CTT) to pheny la l an ine (TTT) in 4/23 h a p l o t y p e 1-mutan t genes (L249F, see Fig. I A ) . The L249F m u t a t i o n was not found in 10 hap lo type 1-normal alleles or in 38 non -hap lo type -1 mutan t genes (P < 0.01).

Fig. 1. A Sequence analysis of the L249F mutation. N, Normal se- quence; M, mutant sequence. The mutant nucleotide is indicated by an asterisk (*). B Pedigree of a family carrying the L249F muta- tion and genotype analysis of PCR-amplified genomic DNA using ASO probes. Haplotype and genotype analysis at codon 249 are presented. The mutant haplotype is indicated by an asterisk (*). N, Normal allele

In order to study the segregation of the L249F muta- tion in affected families, the amplified exon 7 was hy- bridized with either a normal or a mutant ASO probe. Figure 1B shows that the proband (II2) was homozygous for the mutant genotype while her affected mother (I2), her affected brother ( I l l ) , and her healthy father (I1), were all heterozygous for the L249F mutation.

Four mutant haplotype 1 alleles were associated with the R261Q mutation first described by Abadie et al. (1989), but this genotype was also associated with mu- tant haplotype 2 in our series.

Discuss ion

Using a full-length human P A H cDNA sequence as a probe, extensive RFLPs have been identified at the hu- man P A H locus and over 50 RFLP haplotypes have been catalogued (Woo et al. 1983; Woo 1988). Interestingly, most PKU alleles are associated with only four haplo- types in Caucasians, namely haplotypes 1-4 (Chakra- borty et al. 1987). Haplotype 1 is prevalent among mu- tant, as well as normal, alleles throughout Europe but

71

Table 3. Distribution of mutant RFLP haplotype 1 at the PAH locus in Europe (%)

Region Nor- Mu- Reference mal tant

Northern Denmark 32 26 Norway 28 21 Sweden 31 15

Western France 27 31 Germany 25 24

Scotland 32 Switzerland 38

Eastern Czechoslovakia 10 Hungary 32

Southern Italy Portugal Turkey

30 50

Chakraborty et al. (1987) Apold et al. (1990) Svensson et al. (1991)

Rey et al. (1988) Riess et al. (1988) Lichter Konecki et al. (1988b) Aulhela-Scholz et al. (1988) Sullivan et al. (1989) Sullivan et al. (1989)

0 Daiger et al. (1989) 12 Daiger et al. (1989)

29 40 Dianzani et al. (1990) 30 56 Caillaud et al. (1991) 13 28 Lichter Konecki et al. (1989)

the frequency of the mutant haplotype 1 is very different in various European regions. Hitherto, the frequency of mutant haplotype 1 ranged from a low of 0-10% in east- ern Europe to a high of 50% in Switzerland (Table 3) Here, we report a frequency of 56% for mutant haplo- type 1 in Portugal. This is, to our knowledge, the highest frequency for mutant haplotype 1 in all studies reported to date (Table 3). The prevalence of this mutant haplo- type in Portugal approached the record figures for mu- tant haplotype 2 in eastern Europe (54% and 70% for Hungary and Czechoslovakia, respectively, Daiger et al. 1989).

Since several PKU mutations have been described on the background of mutant haplotype 1 genes, we first screened mutant haplotype 1 alleles for the R261Q, R252W and E280K mutations (Abadie et al. 1989; Lyon- net et al. 1989). The E261Q mutation, which accounts for 36% of mutant haplotype 1 genes in Mediterranean countries (Abadie et al. 1989), only accounted for 17% of haplotype 1 PKU alleles in Portugal. Similarly, the L249F mutation, first reported here in association with mutant haplotype 1 at the P A H locus ( P < 0.01), only accounted for 17% of mutant haplotype 1 genes in Por- tugal. Taken together, the R261Q and L249F mutations accounted for only one-third of all mutant haplotype 1 genes in that country. It appears, therefore, that despite strong linkage disequilibrium between PKU and RFLP haplotype i at the PAH locus, this mutant haplotype was genotypically heterogeneous in Portugal. Several mutant genotypes, including E261Q, L249F and the still unknown mutations associated with mutant haplotype 1, might have contributed to the prevalence of this particular RFLP haplotype and to the general incidence of PKU in Portugal (1/14500).

The frequency of the other mutant haplotypes was consistently and markedly reduced. Indeed, haplotypes

72

2 - 4 which account for m o r e than 35% of m u t a n t P A H genes in F rance (Rey et al. 1988) and a p p r o x i m a t e l y 60% of m u t a n t al le les in n o r t h e r n E u r o p e (Da ige r et al. 1989) only accoun ted for 12% of m u t a n t genes in Por- tugal (P < 0.01). In keep ing with this, the R 4 0 8 W and IVS12 muta t ions , a s soc ia ted with m u t a n t hap lo types 2 and 3 respec t ive ly ( D i L e l l a et al. 1986, 1987), were al- mos t to ta l ly absen t in Por tuga l , and the AI94 m u t a t i o n or ig ina l ly desc r ibed in this coun t ry was only found twice in our ser ies (Ca i l l aud et al. 1991). Acco rd ing ly , e ight of the mu ta t i ons r e p o r t e d to da te accoun ted for only one- four th (24 .5%) of all P K U al leles in Por tuga l .

In conclus ion , it is difficult to dec ide at the p re sen t t ime whe the r a few u n k n o w n muta t ions , poss ib ly associ- a ted with the two m o r e f r equen t mu tan t hap lo type s at the P A H locus (hap lo type 1, 56%, P < 0.05; h a p l o t y p e 9, 17%, P < 0.05), account for the o the r m u t a n t geno- types (75%) or w h e t h e r a cons ide rab le d ivers i ty of muta- t ions con t r ibu te to P K U in Por tuga l , as p rev ious ly found in M e d i t e r r a n e a n count r ies (Be r the lon et al. 1991). Fu- ture s tudies d e d i c a t e d to the mo lecu l a r genet ics of P K U in Por tuga l , and in South A m e r i c a n popu la t i ons r e l a t ed h is tor ica l ly and e thnica l ly to Por tuga l , will ce r ta in ly shed more l ight on the origins of the P K U muta t ions in that count ry .

Acknowledgements'. We thank Monique Poussi~re and Alan Strick- land for their help in preparing this manuscript.

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