Proc. Natl. Acad. Sci. USAVol. 92, pp. 9667-9671, October 1995Genetics

Genetic heterogeneity in cystinuria: The SLC3A1 gene is linked totype I but not to type III cystinuria

(rBAT gene/neutral and basic amino acid transporter)

MARiA JULLA CALONGE*t, VICTOR VOLPINI*, LUIGI BISCEGLIAt, FERRAN ROUSAUD§, LUISA DE SANCTIST,ERCOLE BECCIAII, LEOPOLDO ZELANTEt, XAVIER TESTARt, ANTONIO ZORZANOt, XAVIER ESTIVILL*,PAOLO GASPARINIt, VIRGINIA NUNES*, AND MANUEL PALACfNt***Departament de Genetica Molecular, Institut de Recerca Oncol6gica, Hospital Duran i Reynals, Autovia de Castelldefels, Km 2,7, L'Hospitalet de Llobregat,Barcelona 08907, Spain; tDepartament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda Diagonal 645, Barcelona 08028, Spain;tServizio di Genetica Medica, "Casa Sollievo della Sofferenza," and "lDivision di Urologia, "Casa Sollievo della Sofferenza," San Giovanni Rotondo, Foglia, Italy;§Servicio de Nefrologia Institut de Urologia, Nefrologia i Andrologia, Fundaci6n Puigvert, Cartagena 340, Barcelona 08025, Spain; and lIstituto di ClinicaPediatrica, Universita degli Studi di Torino, Piazza Polonia 94, Torino 10126, Italy

Communicated by Leon E. Rosenberg, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ, June 5, 1995 (received for reviewJanuary 31, 1995)

ABSTRACT Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described(I, II, and Ill). An amino acid transporter gene, SLC3A1(formerly rBA7), was found to be responsible for this disorder.To assess whether mutations in SLC3A1 are involved indifferent cystinuria phenotypes, linkage with this gene and itsnearest marker (D2S119) was analyzed in 22 families with typeI and/or type Ill cystinuria. Linkage with heterogeneity wasproved (a = 0.45; P < 0.008). Type I/I families showedhomogeneous linkage to SLC3AJ (Zm.. > 3.0 at 0= 0.00; a =1), whereas types I/III and III/III were not linked. Our datasuggest that type I cystinuria is due to mutations in theSLC3A1 gene, whereas another locus is responsible for typeIII. This result establishes genetic heterogeneity for cystin-uria, classically considered as a multiallelic monogenic dis-ease.

Cystinuria is a heritable disorder (MIM no. 220100; MendelianInheritance in Man) of amino acid transport, transmitted as anautosomal recessive trait (1). This disease is one of the mostcommon genetic disorders with an overall prevalence of 1 in7000 (2, 3). Cystinuria is due to the defective transport ofcystine and dibasic amino acids through the epithelial cells ofthe renal tubule and intestinal tract (4). Cystine has a lowsolubility, and its precipitation results in the formation ofcalculi in the urinary tract, which leads to obstruction, infec-tions, and ultimately to renal insufficiency (2).A human kidneycDNA, named rBAT (also D2; the gene is now designatedSLC3A1 for solute carrier family 3), that elicits the transportof cystine, dibasic amino acids, and some zwitterionic aminoacids via a b° +-like transport system in Xenopus oocytes, wasisolated (5, 6). SLC3A1 protein is expressed in the brushborder plasma membrane of both the proximal straight tubulesof the nephron and the small intestine (7, 8) and is consideredto be responsible for reabsorption of cystine and dibasic aminoacids, most probably through a heteroexchange diffusionmechanism of transport with neutral amino acids (9). We haveidentified six cystinuria-specific missense mutations in theSLC3A1 gene (which we term rBAT). The most frequentmutation found, Met-467 -> Thr, reduces the amino acidtransport activity associated with SLC3A1 in Xenopus oocytes,demonstrating that mutations in SLC3AI cause cystinuria (10).Eight additional mutations in SLC3AI have been found byothers (11) and by Gasparini and coworkers (26). The SLC3AJgene was localized to the short arm of chromosome 2 (refs. 6,

10, 12, 13). Pras et al. (14) reported linkage with homogeneitybetween cystinuria and markers D2S119 and D2S177. We havelocalized the SLC3A1 gene, D2S119, and D2S177 to band2pl6.3 by fluorescence in situ hybridization, showing that theSLC3A1 gene maps at the same locus to which cystinuria hasbeen assigned (13).Three types of classic cystinuria have been described (15).

Type I heterozygotes show normal aminoaciduria, whereastype II and III heterozygotes show high or moderate hyper-excretion of cystine and dibasic amino acids, respectively. Incontrast to type I and II homozygotes, type III homozygotesshow a nearly normal increase in cystine plasma levels afteroral cystine administration. These types are thought to be dueto allelism of the same gene (1, 16), although the involvementof two distinct genetic loci for type I and III cystinuria has beensuggested (17). To clarify the role of SLC3A1 in the cystinuriatypes, we performed linkage studies in cystinuria families oftypes I and III with chromosome 2p markers D2S119 andD2S177 and SLC3AI intragenic markers. Our data demon-strate genetic heterogeneity for cystinuria and suggest thattype I cystinuria is due to mutations in the SLC3A1 gene,whereas another as-yet-unknown locus(i) is (are) responsiblefor type III cystinuria.

MATERIALS AND METHODSPedigrees and Diagnosis. Cystinuria carriers were classified

as either type I or III according to the urinary excretion valuesof cystine and dibasic amino acids (12, 16). Amino acid urineexcretion determinations were done in morning or 24-hr urine,as described (18), and corrected per gram of creatinine.Genotyping Studies. Genomic DNA was amplified with

primers for microsatellites D2S119 and D2S177 (Genethondata base, Evry, France). Using deoxyadenosine [y_32p]_triphosphate end-labeled forward primer, 10-,ul PCRs weredone in a 9600 Perkin-Elmer apparatus. Amplification prod-ucts were then electrophoresed in 6% denaturing polyacryl-amide gels (1500 V) that were then transferred, dried, andexposed to radiographic film. The 1347-02 Centre d'Etude duPolymorphisme Humain (Paris) (CEPH) individual was usedas a size standard.Three mutations (Met-467 -> Thr, Met-467 -> Lys, and

Leu-678 -* Pro) (10) and one polymorphism were used asSLC3AI intragenic markers. Screening for the SLC3A1 poly-

Abbreviations: CEPH, Centre d'Etude du Polymorphisme Humain(Paris); lod, logarithm of odds.**To whom reprint requests should be addressed at: Departament deBioquimica i Fisiologia, Unitat de Bioquimica i Biologia Molecular(B), Facultat de Biologia, Universidad de Barcelona, Avda, Diag-onal 645, Barcelona 08028, Spain.

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morphism + 1854 A/G was done by using the mutagenesisprimer P1854 A/G 5'-GAATTGGTACFTAACCITAGT-CT-3' (nt 1877-1855; the mutated nucleotide is indicated inboldface type) and primer P18D 5'-GAGGAATGACAGCC-ACTATG-3' (nt 1719-1738). Amplification products (159 bp)were digested with Bsm Al (Biolabs, Northbrook, IL).

Linkage and Heterogeneity Analyses. Linkage analysis wasdone by using the LINKAGE 5.2 program package (19). Two-point and multipoint logarithm of odds (lod) scores werecalculated with the MLINK and the iLINK programs. Cystinuriawas assumed to be inherited in an autosomal recessive way with100% penetrance; type III was assumed to be partially dom-inant, whereas type I was recessive. A cystinuria allele fre-quency of 0.012 was used (3), and all marker alleles wereconsidered to be equally frequent. Multipoint analyses weredone by down-coding the markers to five alleles. Two consan-guineous loops were taken into account in pedigrees F6 andF42. Approximate 95% confidence limits were calculated byusing the "1 lod down" method. Recombination frequencieswere transformed to map distances by Kosambi's formula (20).The HOMOG and the MTEST programs (21) were used to test fornonallelic heterogeneity using pairwise lod scores betweenD2S119 and cystinuria at seven recombination values (0,0.001,0.05, 0.1, 0.2, 0.3, 0.4).

RESULTS

Phenotypic Classification of Cystinuria Families. Twenty-two cystinuria families (9 Spanish, 12 Italian, and 1 Belgian)have been used in this study (Fig. 1). A total of 155 individuals,including 43 affected, aged 7-49 yr, were analyzed. Affectedstatus was determined on the basis of clinical presentation andon amino acid urine excretion. All but two individuals had ahistory of cystine stones (Fig. 1).

Cystinuria kindreds were assigned as type I/I (seven kin-dreds in Fig. 1) when all the obligate carriers in the familyshowed normal urinary excretion values (Table 1). The averagerange and sum of urinary cystine and dibasic amino acid levelsin the type I heterozygotes ofthis study agree with reportedvalues (16-18, 22). Obligate carriers showing excretion valueshigher than the range of type I heterozygotes for at least oneamino acid and for the sum of urinary cystine plus dibasicamino acids were classified as non-type I heterozygotes. Noneof the 24 non-type I heterozygotes in this study had a urinaryexcretion of cystine and lysine >750 mmol/g of creatinine and4500 mmol/g of creatinine, respectively, and, therefore, theywere classified as type III heterozygotes (Table 1) and not typeII. In addition, all the sum values of urinary cystine plus dibasicamino acids for all these carriers but two were lower than 4000mmol/g of creatinine, the lower limit given for type II het-erozygotes (17). According to this system, seven kindreds wereclassified as type I/III, and four kindreds were considered astype III/III (Fig. 1). Four kindreds remained undefined be-cause the urinary excretion Values for either one or bothparents of the cystinuric patients were unavailable (Fig. 1).Urinary excretion values for homozygotesI/I and III/III andfor compound heterozygotes I/III shown in Table 1 overlap inthe three groups, agreeing with reported data on adult cystin-uric patients (22).

Cystinuria Phenotypes Show Linkage with Heterogeneityfor Chromosome 2p and SLC3AJ Intragenic Markers. Two-point analyses between D2S119, D2S177, and SLC3A1 andcystinuria were done on the basis of a multiallelic model inwhich type I and type III cystinuria were considered asrecessive and partially dominant, respectively. Seven familiesshowed no cosegregation of the disease with alleles at theD2S119 locus (Z < -2 at 0 = 0), suggesting heterogeneity.Homogeneity tests comparing two-point lod scores betweencystinuria and D2S119 microsatellite or between cystinuria andthe SLC3A1 gene were done. Significant results were obtained

F2

00211412 1 1116 2 2

24 2421 2161 61

F9

122121 213243 1 2

1212 211 3 1 3 224141 32

tVI familiesF 31

D0064 12

61 41

1 3 43

F6

2 112 16 11 6 1

33 52 3364 33 92 3322 -- 22 -- 22 -- 2166 4466 83 66 1 1 6 1

35323634 32323239

64646863 61 6161 61

F 39

1IJ02 1 121

bno6395 65

1222 22

F 36

21 12 5

C 1 141

25 22

64 64

F 29Clo

521211 1

65 64 35 6S62 55

1 2 1Z2 1 2 12 11 1 211 1 1 1 1 _ 11 1iI 11

o 15c b 6b b65 64 35 35 36 62 5SS11 22 -- 12 -- - --1I1 1 7 51 51 11 16 1 6

-Type /ill familiesF7 F14 FIS F25 F280 Lao CI Lao1 O OO52 1 4 9217 8495 74 71 9615

I I 1 2 1 1 12 1 1121 2 1 1 124193 33 32 1 132 32 12 1 233

s n o~~tii-e i 6ii ho54 21 24 2 1 97 97 8949 41 7712 1t 12 11 11 12 12 12 12 -- -- 12111223 49 43 49 4 3232 1313 3231 31 3231

14 4242 52 81 822525 526221 -- -- -- -- 1 1 21

13 3414 14 99 949341 3313

Type 1/111 families Type Ill/Ill familiesF33 F34, F3 FS F26 F42

2 1 1 1211 31 9S 32 12 11 75 132 13..--212 1 2 22 1 2 21 1 1 1 2 22 21 12

21 2S 1 2 32 61 IS2 1 2 1 3 113 1 S I16 12 32

S1 Si 21 1 1 1 1 93 93 2 1 2 1 1 1 75 23 21 1 3 1 121 21 12 12 22 22 12 21 12 1 1 2221

252212 13 13 6565 21 21 11 11 22 231213

Unclassified families

21 -- --52 21 51 5413134343 12 16 25 1611

FIG. 1. Cystinuria families analyzed. Pedigrees of the familiesstudied are grouped on the basis of phenotypic classification ofobligate carriers: type I/I, type I/III, typeIII/III, and unclassifiedfamilies. Square symbols, males; circles, females; diagonal lines, de-ceased; double lines, consanguinity. Nonfilled symbols represent in-dividuals with silent urinary phenotype. Filled symbols denote thehomozygous state (i.e., types I/I, I/III, or III/III), and half-filledsymbols denote cystinuria carriers with a moderate urinary phenotype(i.e., cystinunia type III heterozygotes). Question marks (?) showunclassified individuals. Patients without a history of cystine stones areindicated by arrowheads. Numbers under individual symbols representthe haplotypes given by markers D2S177 (upper), SLC3A1 (middle),and D2S119 (lower). A dash indicates noninformative alleles. FamilyF6 has been reported (10).

for the hypothesis of linkage with heterogeneity versus nolinkage, with a = 0.45 at 0 = 0 for both D2S119 and SLC3A1(Table 2).To assess whether the observed genetic heterogeneity can be

accounted for by the different types of cystinuria we used theoption MTEST on disease status and marker D2S119. Familieswere divided into two groups, those having only type I (i.e.,type I/I families) and those having type III (i.e., type I/III andIII/III families). We found significant heterogeneity (X2 =15.49 with one degree of freedom, P < 0.00008) betWeen the

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Table 1. Urinary amino acid excretion by type I or type III obligate carriers and cystinurics type I/I, I/III, or III/III

Cystine, Lysine, Arginine, Ornithine,mmol/g of mmol/g of mmol/g of mmol/g ofcreatinine creatinine creatinine creatinine Sum

Heterozygotes I/+ 81 + 10 230 ± 26 23 + 3 33 + 8 367 + 32(n = 24) (20-176) (38-445) (10-55) (8-48) (76-632)

Heterozygotes III/+ 311 + 34 1677 + 206 135 + 33 120 + 14 2190 + 231(n = 24) (57-718) (480-4110) (17-714) (28-297) (773-5055)

Homozygotes I/I 1215 ± 285 7118 + 1079 4057 + 684 1650 + 363 13,653 ± 2191(n = 14) (261-3915) (1890-15,600) (165-8194) (21-3800) (4150-31,600)

Compound heterozygotes I/III 1420 + 423 5593 ± 727 2022 ± 551 1223 + 226 10,258 + 1804(n = 13) (240-5556) (1640-11,418) (13-7230) (11-2880) (1900-25,900)

Homozygotes III/III 1354 ± 328 4889 + 641 3224 ± 820 1817 ± 352 11,284 ± 1650(n = 6) (490-2660) (2510-6530) (1950-7270) (920-2990) (7130-17,900)The SEM is given for each average value; n stands for the number of individuals analyzed. Highest and lowest excretion values found in each

group are shown in parentheses. The upper limits of normal urine amino acid excretion values, expressed in ,umol/g of creatinine, are as follows:<176 for cystine, -60 for arginine, s586 for lysine, and <60 for ornithine.

two subgroups, suggesting that the genetic heterogeneity ofcystinuria is related to its several phenotypes.

Cystinuria Phenotype I/I Shows Homogeneous Linkage tothe SLC3AI Cystinuria Locus. In families I/I two-point anal-yses were done between the SLC3AI gene, D2S1l9, D2S177,and cystinuria. Linkage was significant for D2S119 andSLC3A1 and positive, but not significant, forD2S177 (Table 3).The 1-lod-unit support interval for D2S119 ranged from 0 to0.086. The homogeneity test using cystinuria and markerD2S119 showed homogeneity for all these type I/I families (a= 1 at 0 = 0).

Multipoint analysis between D2S177, D2S119, and cystin-uria type I/I yielded a lod score of 5.7 with a genetic distanceof 0 cM between cystinuria and D2S119 and provided conclu-sive evidence that the SLC3A1 cystinuria locus maps centro-meric to D2S1 77. However, it was not possible to localize thislocus either centromeric or telomeric with respect to D2S119.By using unclassified cystinuric families not further used in thisstudy the genetic distance (for 19 informative families) be-tween SLC3A1 and D2S119 was 0 cM withZ = 9.34 (1-lod-unitsupport interval of 0-4.8 cM) and between SLC3A1 andD2S177 was 9.7 cM with Z = 3.24. Two-point analysis betweenD2S119 and D2S177 in 25 informative families gave a recom-bination fraction of 0 = 0.081 with Z = 10.5 (1-lod downinterval of 0 = 0.036-0.155). Multipoint analysis, using 19informative families, with D2S177, D2S119, and SLC3A1intragenic markers shows, as for the SLC3A1 cystinuria locusin type I/I families, that the SLC3A1 gene maps centromericto D2S177 with odds 1:105 in favor of close proximity toD2S119. Again, the location ofSLC3A1 with respect toD2S119could not be assessed because no recombination events be-tween these two loci were found. The regional genetic map of

Table 2. Locus heterogeneity analysis among 22 cystinuria families

X2Components of x2 df D2S119 SLC3AIH2 vs. Hi 1 9.72 6.48

(heterogeneity) (P < 0.002) (P < 0.011)Hi vs. HO 1 1.46 3.56

(linkage) (P < 0.226) (P < 0.059)H2 vs. HO 2 9.72 7.41

(total) (P < 0.008) (P < 0.024)Data are calculated with the HOMOG program. HO is the null

hypothesis of no linkage; Hi is the hypothesis of linkage withhomogeneity; H2 is the hypothesis of linkage with heterogeneity. ForD2S119 and SLC3A1 loci a = 0.45 and 0 = 0. a is the proportion oflinked families at the specific recombination fraction. df, Degrees offreedom. Data were obtained from 22 and 13 informative familiesanalyzed for D2S119 and SLC3AJ loci, respectively.

the cystinuria SLC3A1 locus, based on these results, is shownin Fig. 2.

Cystinuria Phenotypes 1/111 and III/III Are Not Linked tothe SLC3A1 Locus. Two-point analysis at 0 = 0.001 (1 unitsupport limit given by the HOMOG test for families linked to theSLC3AJ cystinuria locus) between the cystinuria phenotypeand locus D2S119 for families type I/III and type III/III isshown in Table 4. The total lod score for this group of familieswas << -2, demonstrating no linkage between cystinuria andD2S1l9. Four of seven type I/III families (F7, F25, F28, andF33) and two of four type III/III families (F5 and F42),representing >50% of these families, showed Ze=o.oo1 < -2(linkage conditional probability s 0.006) and are thereforeconsidered not linked to D2S119. When exclusion is consid-ered at a less stringent recombination fraction (0 = 0.048; 1 loddown support limit between D2S119 and the SLC3A1 gene),two families, F7 and F42, were still excluded. Lack of infor-mativity precludes the demonstration of genetic heterogeneitywithin the 11 families with type III cystinuria (data not shown).An example of the lack of cosegregation between the SLC3A1gene and cystinuria type IIJ/III is shown in a consanguineousfamily (F42) with three affected individuals (Fig. 3). Exclusionof linkage between SLC3A1 and cystinuria in this family is alsosupported because SLC3A1 homozygosity would be expectedin the affected members due to consanguinity. Among the fourunclassified families, F35 has type I and type III heterozygotes.However, the only affected individual in the family cannot beclassified because his phenotype could be either I/I, I/III, orI/II. In any case, the cystinuria phenotype was excluded fromD2S119 linkage with Zo=o.001 = -8.50.To assess whether type I or type III was responsible for the

linkage exclusion in the four type 11fr families we analyzedparental chromosomes. In the three informative families car-rying type I (F7, F25, and F28) three recombinants of seveninformative meiosis between cystinuria and D2S119 wereidentified (Fig. 1). Moreover, there was no cosegregationbetween SLC3A1 intragenic markers and type I cystinuria infamily F7 (Fig. 1). For type III, four recombinants of 12informative meiosis between cystinuria and D2S119 wereidentified in families F5, F25, F28, and F33, and there was nocosegregation between SLC3A1 intragenic markers and the

Table 3. Two-point lod scores between cystinuria andchromosome 2p markers and SLC3AILocus 0.00 0.01 0.05 0.10 0.20 0.30 0.40 Omax Zmax

D2S177 -o 1.13 2.05 2.05 1.52 0.83 0.24 0.07 2.10SLC3A1 3.50 3.42 3.09 2.67 1.81 0.96 0.28 0.00 3.50D2S119 4.22 4.11 3.64 3.07 1.96 0.97 0.26 0.00 4.22

Data were obtained from seven informative families forD2S177 andD2SI19, and four informative families for SLC3AL.

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tel

D2S177

L8 cM

W

+I- D2SI19

cen

FIG. 2. Genetic regional map of the SLC3A1 (rBAT) cystinurialocus. Schematic representation of the short arm of chromosome 2.The region shown for location of the SLC3AI gene represents the 1 loddown support interval (4.8 cM). The marker order of D2S119 andD2S177 is taken from CEPH data (23). Assignment of the SLC3A1gene and loci D2S119 and D2S177 to G-band 2pl6.3 has been reported(13). The D2S119-D2S177 genetic distance reported here (8.2 cM) isbetween those reported by CEPH (5 cM) and by Pras and coworkers(12 cM) (14, 23).

disease in family F28 (Fig. 1). This result demonstrated thateither type I or type III could be responsible for exclusion fromlinkage in cystinuria type I/III families.

DISCUSSIONWe have demonstrated that cystinuria is a heterogeneousgenetic disease. We have shown that type I/I is linked to theSLC3A1 gene (rBAT cystinuria locus). We did not find linkagebetween cystinuria and D2S119 or SLC3A1 for the familiesstudied with cystinuria type III (i.e., type I/III and III/IIIfamilies). Our data strongly suggest that other loci are respon-sible for type III cystinuria. This result clearly contrasts with

Table 4. Exclusion analysis in type I/III and III/IIIcystinuria families

Conditionalprobability of

Family Type linked type Z*

F7 I/III 0.000 -8.99 (excluded)F14 I/III 0.620 0.30F15 I/III 0.620 0.30F25 I/III 0.006 -2.09 (excluded)F28 I/III 0.000 -5.64 (excluded)F33 I/III 0.003 -2.39 (excluded)F34 I/III 0.620 0.30F3 III/III 0.766 0.60F5 III/III 0.000 -4.79 (excluded)F26 III/III 0.729 0.51F42 111/III 0.000 -6.89 (excluded)

Lod score value between the cystinuria phenotype and locusD2S119at 0 = 0.001, which corresponds to the upper 1-unit-support limit(95%) in the homogeneity test. Exclusion has been determined whenZo.ooi values were < -2.00.

the previous view of cystinuria as a monogenic disorder due tomutations in a single gene.The SLC3A1 cystinuria locus has been mapped genetically

between D2S119 and D2S177 and physically to region 2pi6.3(13, 14). Localization of the cystinuria locus 7 cM telomeric tomarker D2S119 has been reported (14). In the present study,the SLC3A1 cystinuria locus has been mapped much closer toD2S119 (0-4.8 cM, 95% support interval). The reasons for thisdiscrepancy are not clear at present, but additional phenotypicinformation on the families studied in the previous report mayhelp clarify this.Rosenberg and coworkers (15) observed families in which

compound heterozygotes (I/II, I/III, or II/III) had full-blowncystinuria. These findings have been explained by allelism ofone gene responsible for the three cystinuria types (1). Incontrast to this, Goodyer and coworkers (17) observed ex-tremely high urinary cystine levels in homozygous (I/I) chil-dren whose parents were silent carriers, whereas cystine ex-cretion by infants of parents with dissimilar phenotypes (silentfor type I and moderately elevated range for type III) waslower. This observation suggested that genetic complementa-

l 2 1 3G A A G1 2 32

D2S 177+1 854 A/G02S 119

Ladder

154pb s |

134pb _

CssC (20-1 76)Lys (38-445)Arg (10-55)Orn (8-48)Sum (76-632)

23 21 13 1 1

AG AA GG GA22 23 1 2 1 3

187 255 674 1,284923 782 6,530 2,51123 180 2,360 2,47263 107 1,650 1,623

1,196 1,324 11,214 7,890

1,1425,2102,333916

9,601

3171,290

47100

1,754

FIG. 3. Cystinuria type III/III family is notlinked to the SLC3A1 cystinuria locus. Pedigree

_ +1 854 A of family F42. Filled and half-filled symbols rep-resent cystinuric patients and carriers, respec-tively. HIaplotypes for D2S177, a SLC3AI genepolymorphism (+1854 A/G), and D2S119 are

854 indicated below the symbols. The gel shows no+ 4 G cosegregation between SLC3A1 alleles andcystinuria. Amino acid excretion values (mmol/gof creatinine) for cystine (CssC), arginine (Arg),lysine (Lys), ornithine (Orn), and the sum ofurinary cystine and dibasic amino acids in thefamily members are displayed below. Range ofvalues for the silent carriers are given in paren-theses.

2 p

25.2

24.3

24.1

23.222.322.12116.3

16.216.1

14

13.2

12

D2S 177+1 854 A/GD2S 119

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tion may be occurring between nonallelic cystinuria genes. Ourstudy demonstrates that cystinuria is genetically heterogeneousand that the SLC3AJ gene is responsible for type I/I cystinuria,whereas other loci could be responsible for type III cystinuria.Involvement of the SLC3A1 gene in type III cystinuria cannotbe completely ruled out due to low informativeness of some ofthe type I/III or type III/III pedigrees studied. Furthermore,mutations in the SLC3A1 gene have only been found in typeI/I alleles (26). On the other hand, the type I/III families inthe present study excluded from having linkage to SLC3A1showed no cosegregation between the SLC3A1 cystinuria locusand either type I or type III cystinuria. This result suggests thatother loci could also be responsible for cystinuria type I. TypeI heterozygotes in type I/Ill-excluded families have the upperrange of excretion values for type I heterozygotes, whereassome of the type III heterozygotes in type I/III-nonexcludedfamilies have the lower range of urinary excretion values fortype III heterozygotes (15, 17, 22). This result suggests thatcarriers of mutations in both the SLC3A1 gene and at anotherlocus could result in a partially overlapping phenotype ofurinary excretion of cystine and dibasic amino acids. Type IIIhomozygotes, but not type I homozygotes, show a nearlynormal increase in cystine plasma levels after cystine oralloading (15). Performance of this test in type I/III and typeIII/III patients might clarify this apparent overlap of pheno-types.A previous linkage study found cystinuria to be a homoge-

neous disorder (14). Our data demonstrate that cystinuria isheterogeneous. On the basis of the results presented here wepropose that different genes are involved in different pheno-types of cystinuria. Type I cystinuria is chiefly associated withthe SLC3A1 gene, whereas type III cystinuria is associated withan undefined locus. This other locus (i) could also be respon-sible for the phenotype seen in some of the type I heterozy-gotes with higher excretion values. However, our data do notdemonstrate or refute the complementation model suggestedby Goodyer and coworkers (17). It would be necessary to studyinformative type I/III pedigrees in which type I is due toSLC3A1 and type III is due to another gene, in the sameindividual, to ascertain this model.The other gene(s) responsible for cystinuria remains to be

found. Several strategies could be used to identify this newgene(s): (i) A positional cloning approach in families that showno linkage to SLC3A1. 14q22 and 20p13 might be the firstchromosomal candidate regions to study, as these regions areinvolved in a de novo translocation found in a compoundheterozygous cystinuric child (24). (ii) An alternative strategywould be to study the candidate genes implicated in thereabsorption of cystine and dibasic amino acids. For instance,preliminary results suggest that the SLC3A1 protein is asso-ciated with an unidentified light subunit in renal brush-bordermembranes (J. Chillaron, unpublished results and ref. 25). Theidentification of an additional gene(s) for cystinuria willestablish the genetic basis of the disease, and also help clarifythe physiology of cystine reabsorption, a process not yet wellunderstood.

We thank the families who participated in this study. We thank P.Barcel6, M. Bruno, A. Rousaud, M. Navarro, J. Jaeken, and M.Nebreda for providing us with cystinuric families. We thank M. D.Ramos for DNA extraction. We also thank A. Ponzone, I. Dianzani,M. Pritchard, J. Purroy, and J. Guimera for helpful discussion andcritical reading of the manuscript. This work was supported in part byDirecci6n General de Investigaci6n Cientifica y Tecnica ResearchGrants PB90/0435 and PB93/0738 (to M.P.), by Fundaci6 August Pi

i Sunyer (to V.N.) and by the Institut Catala de la Salut (to V.N. andX.E.), by Sociedad Espaniola de Dialisis y Transplante (to F.R.), fromSpain, and by Ministero Italiano di Sanita Research Grant (to P.G.),by Telethon94 Grant E 083 (to L.Z.), from Italy. M.J.C. is the recipientof a predoctoral fellowship from the Comissi6 Interdepartamental deRecerca i Innovaci6 Tecnologica from Catalonia (Spain).

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