Original Paper 1610118

Eur J Hum Genet 1994;2:24-34

Sandra Kleiman a Origins of Hyperphenylalaninemia Smadar Avigad* Lina Vanagaitea in Israel Aryeh Shmuelevitzb Miriam Davida Randy C. Eisensmithc Nathan BrandA Gerard Schwartzd Françoise Rey* Arnold Munniche Savio L.C. Wooc Yosef Shiloha a Department of Human Genetics, Sackler School of Medicine, Abstract b Department of Middle Eastern and Mutations and polymorphisms at the phenylalanine hydroxy­ African History, Tel Aviv University, Ramat Aviv, Israel; lase (PAH) gene were used to study the genetic diversity of the c Howard Hughes Medical Institute, Jewish and Palestinian Arab populations in Israel. PAH muta­ Department of Cell Biology and tions are responsible for a large variety of hyperphenylalani- Institute of Molecular Genetics, Baylor College of Medicine, Houston, Tex., nemias (HPAs), ranging from the autosomal recessive disease USA; phenylketonuria to various degrees of nonclinical HPA. Sev­ d Child Development Institute, Sheba Medical Center, Tel Hashomer, Israel, enty-two Jewish and 36 Palestinian Arab families with various and HP As, containing 115 affected genotypes, were studied by c Unité de Recherches sur les Handicaps haplotype analysis, screening for previously known PAH le­ Génétique de l’Enfant, INSERM U-12, Hôpital des Enfants-Malades, Paris, sions and a search for novel mutations. Forty-one PAH haplo­ France types were observed in this sample. Four mutations previously identified in Europe (IVS10nt546, R261Q, R408W and RI 58Q) were found, and were associated with the same haplo­ types as in Europe, indicating possible gene flow from Euro­ pean populations into the Jewish and Palestinian gene pools. Of particular interest is a PAH allele with the IVS10nt546 mutation and haplotype 6, that might have originated in Italy KeyWords more than 3,000 years ago and spread during the expansion of Hyperphenylalaninemia the Roman Empire. These results, together with previous Phenylketonuria identification of three PAH mutations unique to Palestinian Phenylalanine hydroxylase Arabs [IVSnt2, Edel( 197-205) and R270S], indicate that the Haplotypes relatively high genetic diversity of the Jewish and Palestinian Mutations populations reflects, in addition to genetic events unique to Jews these communities, some gene flow from neighboring and con­ Palestinian Arabs quering populations.

Received: July 9,1993 Yosef Shiloh ©1994 Revision received: November 11,1993 Department of Human Genetics S. Karger AG, Basel Accepted: November 22,1993 Sackler School of Medicine 1018-4813/94/ Tel Aviv University 0021-0024S5.00/0 Ramat Aviv 69978 (Israel) Introduction [4-7], PAH mutations show strong linkage disequilibrium with specific polymorphic ha­ Characterization of mutant genes associat­ plotypes at the PAH locus [1-3]. Thus, mu­ ed with hereditary diseases is aimed primarily tant PAH genes can be ‘tagged’ with specific at understanding the molecular basis of these mutation-haplotype combinations, which en­ disorders. However, identification of se­ able one to follow their spread in different quence variations associated with normal and populations with great specificity [8]. defective alleles can also contribute to our More than 2 million Israeli newborns have understanding of the mechanisms underlying been screened for HPA since 1960. PAH defi­ the dynamics of gene flow among human pop­ ciencies were identified in some 450 infants, ulations. Alleles marked by mutations or poly­ 30% of them showing PKU and the rest non- morphic haplotypes or a combination of both PKU HPA [9-11; unpubl. data]. Both types of may serve as useful markers to study the inter­ HPA were identified among Jews and Pales­ relationships between various ethnic groups tinian Arabs. These two populations differ and the origins of their genetic diversity. from the European and Asian communities The gene encoding the hepatic enzyme studied thus far, and from each other. The phenylalanine hydroxylase (PAH) has recent­ Jewish population is composed mostly of im­ ly been a focus of such investigations. PAH migrants from a variety of countries, and their catalyzes the irreversible hydroxylation of descendents, while the Palestinian Arab pop­ phenylalanine to tyrosine, thereby opening ulation reflects the migrations and foreign the catabolic pathway of this essential amino conquests that have swept this country since acid. Mutations in this gene cause a wide the time of the Roman Empire. Both ethnic spectrum of disorders collectively called hy- groups are therefore expected to show consid­ perphenylalaninemias (HPAs) [1]. Extreme erable genetic diversity for their small size. HPA is expressed as the autosomal recessive We are tracing the origins of this diversity, disease phenylketonuria (PKU), which causes using as a probe the mutations and polymor­ severe mental retardation. Early diagnosis of phisms of the PAH gene. PKU and subsequent treatment with a low- phenylalanine diet can considerably reduce the brain damage, making newborn screening for HPA a common practice in many coun­ Subjects and Methods tries. Milder HP As have no clinical conse­ Sample Population quences and are usually identified by bio­ The population of individuals affected with HP As chemical tests. in Israel, and their ascertainment, have been described Extensive studies in European, Mediterra­ previously [4, 9-11]. The great majority of HPA cases nean and Asian communities recently dis­ in the Jewish population was identified among Sephar­ dic and Oriental Jews, rather than in Ashkenazi com­ closed a wide variety of PAH mutations un­ munities [9,10], Over a period of 5 years, we collected derlying these conditions [see ref. 2, 3 for peripheral blood samples from 72 Jewish and 36 Pales­ reviews]. Mutations which affect PAH activi­ tinian families with HP As. Since our primary interest ty severely result in PKU in homozygotes or was directed towards PKU, in most of these families (a compound heterozygotes, while non-PKU total of 88) the probands had PKU. In 13 families non- PKU HPA was identified, and in 7 families both con­ HPA is usually caused by compound hetero­ ditions were segregating [see ref. 4 for a detailed zygosity for a mutation of the severe type and description of these families]. Altogether, 115 affected a mutation with a milder effect on the protein genotypes were studied (each of the 7 families with

25 Table 1. Polymorphic haplotypes identified in nor­ both PKU and non-PKU HPA contributed 2 affected mal and mutant PAH genes in 108 studied families genotypes). Among the Jewish families, 65 were of Sephardic or Oriental origin, 5 were Ashkenazi and 2 Haplo­ Jews Palestinian Arabs represented ‘mixed marriages’. The parents were con­ types1 sanguineous in 34 of the 36 Palestinian families and in normal mutant normal mutant 6 of the 72 Jewish families. genes genes Molecular Methods 1 32 292 14 392 2 7 3 1 3 Extraction of genomic DNA from peripheral blood 3 2 3 samples, haplotype analysis, identification of pre­ 4 34 16 14 4 viously known mutations and the search for new muta­ 5 4 3 3 4 tions have been previously described in detail [4, 12- 6 2 21 2 12 16]. 7 6 6 7 8 8 2 9 2 10 4 2 Results ll l 1 12 7 1 4 14 1 Polymorphic Haplotypes at the PAH Locus 15 1 Eight RFLPs conveniently detected by the 18 1 1 full cDNA of PAH [17, 18] were used to con­ 19 1 27 3 1 2 struct polymorphic PAH haplotypes in all the 28 10 4 2 families studied. Forty-one haplotypes were 29 1 observed in the sample population. Their dis­ 32 1 33 2 tribution among normal and mutant PAH 36 1 2 genes is shown in table 1. Complete haplotype 37 2 analysis was not possible in some families, 41 1 2 42 1 2 4 since not all parents were available. In other 43 1 families, heterozygosity of all family members 44 1 for one or two RFLPs and unavailability of 47 1 50 1 additional family members precluded haplo­ 52 2 1 type construction. Hence, the final number of 56 1 haplotyped chromosomes is slightly smaller 57 1 58 1 than expected on the basis of the sample size. 59 1 Except for haplotype 6del which is unique to 61 1 Yemenite Jewish PKU patients [19], all of the 62 1 63 1 haplotypes identified in this study have also 64 1 been observed in European and Mediterra­ 65 1 nean populations [2, 3, 8, 18, 20]. 66 1 6del3 52 Although a considerable variety of haplo­ Total 135 144 63 76 types was found among Jews and Palestinian Arabs, in both groups most of the haplotypes l Haplotype designation is according to Woo [18]. occurred on a single or small number of chro­ 2 Significantly different between Jews and Palestin­ mosomes, with three or four haplotypes pre­ ian Arabs (p < 0.001). 3 A derivative of haplotype 6 containing a deletion dominant. Haplotypes 1, 4 and 6 were abun­ that eliminates the PvuII (b) RFLP [19]. dant among both Jews and Palestinians; ha­ plotype 28 was also frequent among Jews, as

26 Kleiman et al. Hyperphenylalaninemia in Israel Table 2. Geographic origin of mutant PAH genes among Jews

Country Haplotypes of origin 1 2 4 5 6del 6 7 10 12 18 27 28 36 41 64

Algeria 1 1 Tunisia 2 1 Morroco 4 4 16 1 1 2 Egypt 1 Yemen 3 1 52 1 1 2 Iraq 8 1 1 1 Iran 2 1 3 1 Syria 1 Turkey 5 1 1 2 1 Afghanistan 2 1 Azerbaijan 3 1 Kurdistan 1 Poland 2 1 Lithuania 1 Yugoslavia 1 Germany 1 1 Russia 4 Rumania 2 Total 29 3 16 3 52 21 6 2 1 1 1 4 2 2 1

was the ‘Yemenite’ PKU haplotype, 6del. For Point Mutations in the PAH Gene haplotypes other than 6del, %2 analysis indi­ Tables 3 and 4 summarize PAH molecular cated significant difference between frequen­ lesions found to date in the Israeli population, cies among Jews and Palestinian Arabs only their haplotype association and ethnic origins. for mutant haplotype 1. The entire patient population - excluding the Most haplotypes were shared by wild-type Yemenite Jewish PKU patients who are all and mutant alleles, and only two haplotypes homozygous for a deletion of PAH exon 3 [ 19] in Jews and two in Palestinian Arabs were - was screened for the presence of eleven unique to mutant alleles (table 1). Haplotype point mutations previously identified in Eu­ 6 was found almost exclusively on mutant ropean and Mediterranean populations: chromosomes in both groups. The major ha­ R158Q, R252W, R261Q, G272X, S273F, plotypes on mutant alleles among Jews were E280K, P281L, L31 IP, IVS10nt546, R408W 6del, 1, 4, 6 and 7 (table 1). While more than and Y414C [2, 3]. Four of these mutations half of the mutations associated with haplo­ were identified on 50 of the 168 mutant alleles types 4 and 6 originated in North African tested; 35 of the 50 alleles contained the splic­ communities (table 2), mutant haplotype 1 ing defect IVS10nt546 (table 3). Notably, the was found mainly in Jewish patients from haplotype association of these mutations (ta­ Middle Eastern countries. ble 4) was similar in the Israeli and European populations, with the exception of two alleles

27 Table 3. PAH mutations identified in the Israeli population

Mutation1 Location2 Consequence Method of Number of Haplotype association Haplotype References detection3 positive in European and association PAH genes Mediterranean in Israel in Israel4 populations

Mutations previously identified in other populations IVS10nt546 110 cryptic splice RED 35 6, 10,16,24, 36, 43 5,6,36 31,32,36-41 R261Q E7 Arg Gin RED 9 1,2,4,28 1 25,27, 32, 36, 42-48 R408W E12 Arg-»Trp RED 4 1,2,5,34 2 5,28, 30, 32-34, 36, 40,47, 49, 50 R158Q E5 Arg Gin ASO 2 4, 16,28 4 32, 35, 36,47, 48,51,52 Mutations identified and characterized in Israel delE3 E3 deletion SB 52 6del 19 IVS2ntl 12 defective RED 12 7,42 14 splicing E6del( 197-205) E6 deletion GE 2 4 13 R270S E7 Arg -> Ser ASP 4 1 16 S349P5 E10 Ser —> Pro ASO 6 1 4 12,21,22

l The numbers denote positions of PAH residues and following letters denote the original and substituting residues, respectively. 2 E = Exon; I = intron. 3 ASO = Allel-specific oligonucleotide; RED = restriction enzyme digestion; SB = Southern blotting; GE = gel electrophoresis; ASP = allele-specific PCR. 4 Out of 220 mutant PAH genes. 5 This mutation was discovered independently by Forrest et al. [21] and John et al. [22]

Table 4. Association between PAH lesions, polymorphic haplotypes and ethnic origin in the Israeli population

Mutations: IVS10nt546 R261Q R408W R158Q delE3 Haplotypes: 6 5 36 others 1 others 2 others others 6del others

Palestinians 12/12 2/4 0/60 6/39 0/37 1/3 0/73 2/4 0/72 0/0 0/76 Jews Tunisia Morroco 15/16 1/2 Yemen 52/52 Iraq 1/1 2/8 Iran 3/3 1/2 Afghanistan 1/1 Poland 2/2 Yugoslavia 1/1 Total 20/21 0/3 1/2 0/118 3/29 0/116 3/3 0/141 0/144 52/52 0/92

28 Kleiman et al. Hyperphenylalaninemia in Israel carrying the IVS10nt546 mutation on the was studied by screening 17 Moslem Moroc­ background of haplotype 5, found in a Pales­ can patients for the same mutations. The tinian Arab patient. This association suggests IVS10nt546 mutation was found in one pa­ that several mutant PAH alleles among Jews tient, in association with haplotypes 6 and 36, and Palestinians share common origins with and S349P was identified in another one, with similar alleles in these countries. haplotype 4. A search for novel point mutations in the sample population has yielded thus far four mutations: E6del( 197-205) [13] IVS2ntl Discussion [14], R270S [16] and S349P [12]. The first three mutations are unique to Palestinian The variety of polymorphisms and muta­ Arabs and were not reported in any other pop­ tions at the PAH locus makes it a useful tool ulation, while S349P was discovered indepen­ in molecular population genetics. Due to the dently in French-Canadian and European pa­ strong linkage disequilibrium between PAH tients [21, 22], Haplotype association of these mutations and specific haplotypes, it is highly mutations is shown in table 4. probable that alleles carrying the same muta- Two mutations were identified in 20 of 28 tion-haplotype combination are identical in mutant PAH genes among Moroccan Jewish descent. This notion allows one to trace the patients: IYS10nt546, which was associated origin of such alleles back in time and geogra­ mostly with haplotype 6, and on one chromo­ phy, and to uncover mechanisms underlying some with haplotype 36; and S349P, all asso­ the genetic diversity or genetic similarity of ciated with haplotype 4 (table 4). The ori­ different communities. Typical examples are gin of these chromosomes in this community the identification of a single PAH deletion

IVS2ntl E6del(197-205) S349P R270S 7 42 others 5 others 4 others 1 others

8/8 4/4 0/64 2/4 0/72 0/4 0/72 4/39 0/37

2/2 4/4

0/144 0/144 6/16 0/127 0/144

29 responsible for all PKU cases among Yeme­ able genetic variability. But, a small number nite Jews, which was traced back to specific of haplotypes account for the majority of families several hundred years ago [19], and PAH alleles identified. The geographic origins the recent localization of the origins of several of the mutant PAH haplotypes in the Israeli PAH mutations to specific European ethnic Jewish population summarized in table 2 subgroups [8]. show that the majority of HPA cases among The two populations investigated in this Jews in Israel are in families of north African, study are very different from each other: The Middle Eastern and Asian origin. It is note­ Jewish population of Israel is composed most­ worthy that in some cases the same haplotype ly of immigrants who have come from numer­ appears on mutant alleles from countries in ous countries since the beginning of the 20th different parts of the world, e.g., haplotypes 1, century, and their descendents. These people 4, 6 and 7. have ancient roots in this region that date However, haplotypes alone, particularly back to the Jewish Kingdom of the first and the more abundant ones, give only a weak second millenia BC. The spread of the Jewish indication of genetic similarity and can be people around the world following the de­ used to pinpoint genetic relationships be­ struction of the Kingdom and deportation by tween different communities only when the the Romans in the first century AD should associated mutations are identified [26]. Ha­ have resulted in increased genetic diversity plotype 1, for example, was found to harbor at despite their relative genetic seclusion in the least 14 different mutations in Europe [2], It is diaspora. The Palestinian Arab population noteworthy in this regard that mutations com­ originated in Arab tribes that migrated to Pal­ mon to the Israeli and European populations estine from the Arabian Peninsula at the time are associated in most cases with the same of the Roman-Byzantine Empire, and ex­ haplotypes (table 3), indicating possible com­ panded during the Arab-Islamic conquest of mon origins. Three of these mutations - the country. The Palestinian Arabs have been R158Q, R261Q and R408W - are associated residing in this country since that time, living with the dinucleotide CpG, a known hot spot under different rulers and assimilating var­ for mutations [27], and therefore recurrence ious waves of immigrants, including the cru­ of these mutations cannot be ruled out [28, saders and the Turks. 29], However, it seems to us that the similar Haplotype analysis in this study was based haplotype association favors the possibility of on eight RFLPs [17] which served to establish common origin, particularly in the case of the a commonly used haplotype nomenclature R408W/haplotype 2 allele which is rare in the [18], Additional polymorphisms are being Israeli population (table 4). It is not surprising discovered at the PAH locus [23-25], and to see this allele in Jews from eastern Europe their incorporation into the existing haplo­ (table 4), since it has a Balto-Slavic origin and type system should refine this nomenclature accounts for a high proportion of PKU alleles [20]. It should be noted that the subjects stud­ in eastern Europe and Germany [2, 8, 30-35]. ied do not constitute random samples of their It is interesting to note the appearance of this communities, since only families with HP As allele and the R158Q/haplotype 4 combina­ were selected for analysis. A relatively large tion in Palestinians (table 4). Gene flow from number of different haplotypes was found in Europe into the Palestinian gene pool could the Jewish and Palestinian Arab populations have occurred at various periods in history: in relation to their size, reflecting consider­ during the waves of migration from western

30 Kleiman et al. Hyperphenylalaninemia in Israel Fig. 1. Boundaries of the Ro­ man Empire at its height during the 2nd century A.D.

and central Europe (including the Balkan re­ rope during the expansion of the Roman Em­ gion) in the 1 st century BC, during the Roman pire in the first century BC. The regions conquest of the region up to the Arab-Islamic included in that Empire at its height in the conquest in the 7th century AD, and during 2nd century AD (fig. 1) roughly correlate with the Crusades from 1099 AD through the 13th the current prevalence map of this allele. The century. Additionally, Christian pilgrimages appearance of the mutation in Palestinian to the Holy Land were continuous throughout Arabs may reflect the Roman and subsequent the last 20 centuries. Byzantine centuries-long rule in Palestine. Most prominent on the list of mutant PAH The local Christian population assumed the alleles common to Israeli and other popula­ Islamic religion during the Arab conquest in tions is IYS10nt546/haplotype 6 (tables 3, 4). the 7th century, and was totally assimilated It shows a unique and widespread geographic into the Arab population. distribution, with particular prevalence in The predominance of the IVS10nt546/ha- eastern Europe and Turkey, and has also been plotype 6 chromosome among Moroccan reported in Italy, Spain and western Europe Jews is intriguing. The frequency of this chro­ [31, 36-41], Romano et al. [41] detected this mosome in our Moslem sample population is allele in the eastern part of Sicily and conclud­ low (1/34) but, interestingly, the only other ed that the origin was probably Italic, before IVS10nt546 chromosome in the Moslem the 10th century BC. A compilation of the sample carries haplotype 36 found with this geographic locations of the IVS10nt546/ha- mutation in one Jewish patient. Another PAH plotype 6 allele based on this and previous allele found in one Moslem patient and studies leads us to suggest that this allele may among the Jewish patients in Morocco is indeed have originated in Italy before the first S349P/haplotype 4. Jewish-Moslem intermar­ millenium BC, and spread through the Medi­ riages were extremely rare in this country, terranean littoral countries and eastern Eu­ being strictly forbidden by the Jewish religion.

31 A possible source of the mutation in the A search for novel mutations among Pales­ Jewish gene pool might be the conversion to tinian PKU patients has led to the identifica­ Judaism of local populations, including the tion of 3 ‘private’ mutations not shared by Berbers, that took place during the last centu­ other populations (table 3). It is still possible ries of Roman and Byzantine rule in the area, that these mutations did not occur within the before the Arab-Islamic conquest at the end of Palestinian community, and might be com­ the 7th century. mon to other Arab communities in the region The few IVS10nt546/haplotype 6 alleles - a question that can be resolved by screening found in Jews from Asian countries (table 4) PKU patients in neighboring countries. can be attributed to occasional migrations The usefulness of PAH genotypes as popu­ eastward of Jews fleeing religious persecution lation genetic markers should increase consid­ under Roman/Byzantine rule. A similar con­ erably once the entire spectrum of HP A muta­ clusion may be drawn for the R261Q/haplo- tions is revealed and haplotype nomenclature type 1 allele which, however, does not pene­ further refined by additional polymorphisms. trate the Moroccan-Jewish gene pool. We expect these sequence variations to dis­ A clear example of a recurrent mutation in close additional, unknown origins accounting our population is S349P. This mutation was for the genetic diversity of the peoples of our identified independently in an Australian region. PKU patient for whom the haplotype associa­ tion was not reported [21], and in French- Canadian patients in association with haplo­ Acknowledgements type 1 [22], The same mutation was discov­ We thank the staff of the Child Development Insti­ ered independently in our laboratory in Je­ tute at the Sheba Medical Center for their help wish patients of north African extraction, on throughout this study, and the patients and their fami­ the background of haplotype 4 [12]. The re­ lies for their cooperation. We are grateful to Dr. C.R. currence of this mutation is particularly inter­ Scriver and his staff for compiling and distributing the esting because it does not involve a CpG dinu­ PAH Mutation Consortium lists. SLCW is an investi­ gator at the Howard Hughes Medical Institute. This cleotide. study was supported by the US-Israel Binational Science Foundation (BSF).

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34 Kleiman et al. Hyperphenylalaninemia in Israel