Cystinuria genotypes predicted from excretion patterns

American Journal of Medical Genetics 2: 175-190 (1978)

Cystinuria Genotypes Predicted From Excretion Patterns Sally Kelly

Birth Defects Institute, New York State Department of Health, Albany

Genotypes of 17 patients with cystinuria were predicted from data based on excretion rates of the families’ obligate carriers. The methodology differed from that used by other investigators as it did not employ intestinal biopsy studies or loading dose measurements. The Type I form was more common than either Type I1 or Type 111 and frequently occurred in combination to give compound heterozygous genotypes with the Type 111 form.

Key words: cystinuria, phenotypes, genotypes, excretion patterns, obligate carriers

INTRODUCTION

Cystinuria, the recessively inherited stone-forming disease attributed to defects in membrane transport [ 1,2] , is comprised of genetic subtypes. Indistinguishable clinically, the subtypes were first recognized by Harris and colleagues when healthy relatives in certain families were found to be mildly cystinuric [3]. The disease in these families was considered “incompletely recessive,” in contrast to its truly recessive nature in families where carriers did not express the trait [4]. Confirmed by Rosenberg et a1 151, Crawhall, Saunders, and Thompson [6 ] , and later by others, these observations were extended by Rosenberg and his co-workers, who distinguished two subtypes of the incompletely recessive form, Types I1 and 111 [S] , and combinations of subtypes [7,8]. Using more quantitative methods than were available previously, they distinguished between carriers of the respective subtypes by detecting differences in degree of excessive excretion and demonstrated corresponding differences in patients, eg, in extent of amino acid uptake by intestinal tissue [5, 71 and response to loading doses [5]. Later, others also distinguished the incompletely recessive forms or combinations by interpreting data from 24-hour excretion rates of carriers [9, lo ] , satisfying Rosenberg and associates’ criteria [ 1 11 , or by observing loading dose responses in carriers [ 121 .

Received for publication August 3, 1977; revision received January 25, 1978.

Address reprint requests to Sally Kelly, MD, Birth Defects Institute, Division of Laboratories and Research, Empire State Plaza Tower Building, Albany, NY 12201.

0148-7299/78/0202-0175$02.90 0 1978 Alan R. Liss, Inc

176 Kelly

The few data relating to distribution of subtypes suggest the possibility of geo- graphic differences. Twice as many families in England [4], for example, and five times as many in Sweden [13] had recessive forms as had incompletely recessive forms, among 25 and 59 families studied, respectively. Only about half the North American families observed, however, displayed recessive (Type I) phenotypes - 5 of 12 in the United States [5] and 4 of 10 in Canada [ lo] . Conversely, the recessive form was rare in a group of Jewish families of Libyan origin, where it was found just once among 11 families, compared to its occurrence in 6 of 10 non-Libyan Jewish families with the disease [14].

Types I1 and I11 have been found in the United States [5 ,7 ,9] and Germany [12] and the Type I1 form only, in Canada [ 11 ]

and clearly demonstrated that urinary amino acid excretion patterns can be used for presumptive genotype assignments without recourse to intestinal biopsy or amino acid loading studies [7 ,8] . We subtyped the disease in the families of 17 patients with a history of stone formation, whose samples we received for diagnosis or for study of thera- peutic response. We distinguished phenotypes by recognizing differences in cystine and dibasic amino acid excretion in obligate carriers, Rosenberg and associates’ initial criterion. We predicted genotypes, in turn, from the history, pedigree, and family phenotype(s). The series includes the largest number of patients in the United States in whom the disease has been subtyped and provides information on subtype distribution in a typical North American population.

Even fewer data relate to subtype distribution of the incompletely recessive forms:

We enlarged the data base on which Rosenberg and co-workers classified cystinuria

METHODS AND MATERIALS

Excretory patterns of the stone-forming patients were examined for characteristics which might distinguish those of different genotype, although similar analyses have not been discriminative [7, 151. Of greater promise, however, were the patterns of obligate carriers, ie, parents or offspring of stone-forming patients. We examined them for the features Rosenberg and co-workers considered expressive of the Types I, 11, or 111 heterozygous phenotypes [5,7,8] and were able, when they were present, to predict the respective stone-forming patient’s genotype.

All excretory patterns were interpreted from measurements of 24-hour excretion rates of cystine, lysine, arginine, ornithine and the mixed disulfide, homocysteine- cysteine, expressed in milligrams per day [7, 161. Since 24-hour collections may be in- accurate, especially when collected in the home, as many of ours were, patterns were also interpreted from estimates of the concentrations of the respective amino acids, expressed in milligrams per gram creatinine. Cystine rates and concentrations were calculated as the sum of free cystine and the cysteine of the mixed disulfide homocysteine-cysteine. The cysteine of penicillamine-cysteine was included [ 17, 181 in the total cystine excreted by nine patients who were receiving the drug.

from three patients with histories of renal colic. Twelve were men, ranging in age from 6 to 58 years (mean age 25 years) when first sampled for our study. The women patients were from 14 to 49 years old (mean age 26 years).

Similar samples were obtained from the obligate carriers in 15 of the 19 families - from both parents in 11 families, one parent only in two families (one with offspring and

Urine was collected from 18 stone-forming patients, including two pairs of sibs, and

Cystinuria Genotyping 177

sibs and the other with sibs only) and from offspring only in two families. Samples from sibs in 10 families and from more distant relatives in two families were also tested. Samples from obligate carriers in four families were not available.

The collections were preserved with thymol or 0.1 N HCl. They may or may not have been refrigerated before transport, and upon receipt in the laboratory they were dis- pensed as aliquots and frozen at -20°C before processing. The failure to acidify before refrigeration may have reduced the cystine content of collections from stone-forming patients, as samples with an initial concentration of 500 mg/liter (an amount close to the mean excreted by stone-forming patients) and acidities of pH 5.4 contained 18% less cystine after 24 hours at 4°C. We are assured that the carriers' samples (where rates and concentrations were critical to our thesis) were little changed, on the other hand, if non- acidified samples were in fact refrigerated during collection, since the loss when the cystine concentration was 80 mg/liter, an amount slightly greater than that excreted by most of our carriers, was only 2%.

Samples from the patients were screened for cystinuria by nitroprusside [19] and one-dimensional paper chromatography [20] before further study.

The excretion rates were calculated from estimates of the neutral, basic, and acidic amino acids measured on a Beckman 120C analyzer. Neutral and acidic amino acids were eluted from PA28 resin with 0.2 N citrate buffers of pH 3.20 for three hours and pH 4.30 for three hours, at 30°C for 1.75 hours and 55OC for the remaining time. Basic amino acids were eluted from PA35 resin with 0.38N citrate buffer of pH 4.29 for three hours and 0.35 N buffer of pH 5.26 for 2.5 hours, at 33°C for three hours and 55°C for the remaining time. The mixed disulfide, cysteine-homocysteine, was resolved in a rechromatog- raphy of the sample with the neutral and acidic buffer change postponed for an addi- tional 150 minutes.

per gram of creatinine; creatinine in acidified aliquots, heated to 120°C for 30 minutes at 15 lb pressure to promote conversion of creatine to creatinine, was measured by an automatic method [21].

Rates and concentrations in samples from stone-forming patients were compared with those of 16 adult controls (10 men) of 17-50 years of age. Data from the carriers and other relatives were matched with data from controls of the appropriate age, eg, 4 newborns, 3 weeks old or less; 24 infants (16 boys) 20 months old or less; 15 children (8 boys) less than 8 years old; 19 children (10 boys) 8-16 years old; and the 16 adult controls. All controls were patients suspected of having aminoacidopathies not related to cystinuria or cystinuria-provoking disease and whose excretory rates of cystine and the dibasic amino acids were not greater than published normal rates when measured by ion exchange column chromatography 122,231 (Table I).

Concentrations of cystine and the dibasic amino acids were expressed in milligram

RESULTS

All the stone-forming patients excreted cystine and the dibasic amino acids at rates in excess of normal rates. Cystine excretion rates varied from 112 mg/day, the volume excreted by a patient in end-state kidney failure following repeated stone-formation and nephrectomy (patient P), to a maximum of 1,212 mg/day, excreted by a 26-year-old man, patient E. Mean and median rates for the group were 575 and 545 mg/day. Lysine

178 Kelly

TABLE I. Maximum Excretion Rates (a) and Concentrations (b) of Cystine and Dibasic Amino Acids (mg/day and mg/gm creatinine, respectively) by Noncystinuric Children and Adult Controls Measured in Our Laboratory

Age Cystine Lysine Arginine Ornithine (yr) Number a b a b a b a b

< 2 24 5 30 9 116 1 6 1 29 2-1 15 6 12 28 41 2 4 2 3 8-16 19 18 33 33 28 3 8 3 5

11- 16 28 31 50 44 6 4 3 3

was excreted at rates from two to five times higher than cystine, for the most part, ranging from 296 (patient P) to 4,870 mg/day (patient K). Mean and median rates of lysine excretion were 1,380 and 1,165 mg/day, respectively. Arginine was excreted at rates similar to cystine, on the whole, ranging from 50 (J) to 1,842 mg/day (R); mean and median rates were 669 and 654 mg/day, respectively. Ornithine was excreted by most patients at lower rates, however, ranging from 43 (S) to 1,579 mg/day (E), with mean and median rates of 333 and 294 mg/day. No rates were modal.

Cystine concentrations ranged from 31 mg/gm creatinine (B, a 14-year-old girl) to 1,075 mg/gm Cr (D, a 6-year-old boy). Mean and median concentrations of cystine were 428 and 390 mg/gm Cr, respectively. Lysine concentrations ranged from 362 (P) to 2,790 mg/gm Cr (R), with mean and median concentrations of 1,030 and 1,010 mg/gm Cr, respectively. The arginine excretion range resembled that of cystine, from 31 (J) to 1,640 (D), with mean and median concentrations of 524 and 467 mg/gm Cr, respectively. Ornithine concentrations ranged from 33 (S) to 676 (D), with mean and median concentrations of 222 and 193 mg/gm creatinine, respectively.

rates and concentrations, for the most part (Table 11). The women’s lower excretory rate of lysine, however, was significant at the 5% level.

Men and women patients excreted cystine and the dibasic amino acids at similar

TABLE 11. Mean Rates (mg/day) and Concentrations (mg/gm creatinine) of Cystine and the Dibasic Amino Acids Excreted bv Men and Women Patients

Men (1 2) Women (9) Amino acid Mean SE Mean SE

Cystine mg/day mg/gm creatinine

mg/day mg/gm creatinine

Arginine mg/day

Lysine

mg/gm creatinine Ornithine

mg/day mg/gm creatinine

587 98 496 100 427 40 431 64

1,868 385 1,024 115 1,012 74 946 103

723 141 684 102 544 72 481 82

448 128 238 59 237 24 191 34

SE - standand error of the mean.


The interindividual variation in cystine concentration (milligrams per gram creatinine) was not significantly different from the intraindividual variation when we compared excretion in the 10 patients from whom we had obtained successive samples over a period of years. The difference, expressed by a variance ratio of 1.07, was not significant at the 5% point, when entered in F tables, employing 9 and 22 degrees of freedom, respectively. The interindividual variation was greater, however, when we limited the comparisons to excretion by the five patients from whom successive samples had been obtained within single years. The difference, expressed by a variance ratio of 9.99, was significant at the 1% point, employing 6 and 7 degrees of freedom. Finally, the variation in successive samples obtained within a single year was, in one patient, significantly less than in samples obtained in different years (variance ratio F was signfi- cant at the 0.1% point, with 4 and 2 degrees of freedom), whereas, in two other patients for whom we had the appropriate samples, the difference in variation in the two kinds of successive samples was not significant.

We examined the data for clues to the possibility that age of patients at the time of sampling may have contributed to the variation. We were unable to discern differences with age, however, when we compared cystine excretion in the same patient at different ages. The variation in successive samples, as described above, may have masked changes with age, if indeed there were any. Nor were there trends, for the most part, when we ranked concentrations of amino acids with age of patient at the time of sampling, that is, the differences between the means in comparisons of cystine, arginine, and ornithine concentrations were not significant. Older women, however, excreted slightly greater, and younger men clearly greater, concentrations of lysine (Table 111).

The data suggested, on the other hand, that penicillamine treatment affected the amount of cystine excreted. Cystine concentrations were greater, for example, before and after, rathsr than during, the therapy in the three patients for whom we had appropriate samples. The mean of the differences with and without treatment (1 19, 365, and 389 mg cystine per gram creatinine) was significant at the 5% level. Furthermore, the mean concentration of cystine in 21 samples from patients not receiving penicillamine was higher than in 22 samples from patients receiving the drug, a difference significant at the 5% level. Additionally, when the 43 samples were ranked in order of cystine concentration, those in the upper quarter of the array, containing 565-1,075 mg cystine per gram creatinine, were all from patients not receiving penicillamine at the time of sampling,

TABLE 111. Concentrations of Cystine and Dibasic Amino Acids After Being Ranked With Age At Time of Sampling, Expressed as Mean Concentration (mg/gm creatinine) in Samples From Men and Women Patients Younger (-) or Older (+) Than the Respective Median Ages of 23.5 and 29 Years

Men Women Amino acid - + Diff. - + Diff.

Cystine 407 420 13 45 1 426 25 Lysine 1,731 908 823a 781 1,155 374b Arginine 616 472 144 382 597 215 Ornithine 280 209 71 158 219 61

“Significant at the 1% level. bignificant at between the 5 and 10% levels.

180 Kelly

whereas only four samples from the patients receiving penicillamine contained cystine in as high a range as 500-560 mg per gram creatinine.

Thus, like investigators before us [4,7], we did not discern differences in the excretion patterns of stone-forming patients that would enable us to predict genotypes. Family studies, on the other hand, as described below, revealed a series of patterns in obligate carriers which resembled those in the families Harris, and, later, Rosenberg employed to predict the respective stone-former’s genotype.

One or more of the obligate carriers in 13 families excreted normal amounts of cystine and the dibasic amino acids, we considered the patterns expressive of the truly recessive or Type I heterozygous phenotype. One or more carriers in six families excreted the amino acids in slightly greater amounts (up to twice the normal range); we assigned the Type 111 form of the incompletely recessive heterozygous phenotype. Carriers in two families excreted large amounts of cystine and lysine (9- 15 times the normal range), but less than the amounts excreted by most of the stone-forming patients, and they did not themselves form stones ; these were assigned the Type I1 incompletely recessive heterozygous phenotype (Table IVA).

Some obligate carriers excreted more lysine in proportion to cystine than other carriers. Recognized earlier by Rosenberg et a1 [ 51 and Crawhall and co-workers [6, 171 , the anomaly was designated in our carriers as a “lysine variant” of the respective type if the lysine was at least four times greater than the cystine excretion. Four of the anomalous phenotypes occurred in children, two of whom, sons of patient L, displayed more expressive phenotypes when older. Two other “lysine variants” were found in mothers, one who excreted the other amino acids at normal rates, ie, at rates consistent with the Type I heterozygous phenotype, and the other who excreted at rates consistent with the Type 111 form (Table IVB).

The excretory data used in assigning the phenotypes above were expressed as rates (milligrams per day) and concentrations (milligrams per gram creatinine). When there was a discrepancy between the assignments because of the coefficient used, as there was in assigning the phenotype of C’s 4-year-old daughter, who excreted at normal rates (Type I) but in concentrations higher than normal for her age (Type III), the more abnormal phenotype was assigned.

children. When excretion in successive samples obtained in different years was compared, both rates and concentrations of the amino acids increased in the children who ultimately expressed the Type I1 heterozygous phenotypes (Table V). We had difficulty, therefore, in distinguishing between the Types I1 and I11 heterozygous phenotypes in young children and deferred the assignments, when questionable, until they were older. In particular, the phenotype in patient L’s younger son, which resembled the Type 111 heterozygous form at 6 years of age, changed, by age 12, t o the Type I1 phenotype already expressed by his older sibs and maternal grandfather. The phenotype of L‘s older son, first assigned as Type IIIlys at 10 years of age, shifted to the more abnormal Type I1 pattern by age 15.

When ranked with age, furthermore, the rates excreted by older children expressing the Type I1 heterozygous phenotype were significantly greater than those excreted by the younger children (Table VI). The mean concentrations of cystine and lysine excreted by the older children of this phenotype were also significantly greater.

We paid particular attention to the expression of phenotypes in obligate carrier


A trend with age was not apparent, however, in the incompletely recessive carrier children of Type 111 phenotype. The rates in the obligate carrier daughters of patient H, for example, did not change with age (Table V). When ranked with age, furthermore, the rates and concentrations in older children expressing this phenotype were not significantly greater, for the most part, than those excreted by younger children (Table VII). The comparisons in this group may be premature, however, as the children were uni- formly younger during the sampling period (median age 5.5 years) than those expressing Type I1 phenotypes (median age 10 years). Excretion rates and concentrations, furthermore, were considerably lower for the age than in their Type I1 counterparts (Tables VI and VII).

Type I1 heterozygous phenotypes were assigned when excretion rates and concentrations were considerably higher than in obligate Type 111 carriers, but less than in stone- forming patients. The relative arginine excretion was an especially valuable clue in distinguishing this phenotype : It was disproportionately less than the arginine accompanying the gross excretion of cystine and lysine by stone-forming patients or “biochemically homozygous” healthy relatives, but distinctly greater than in carriers expressing Type 111 heterozygous phenotypes. Indeed, the relatively low arginine was a critical factor in pre- dicting a heterozygous genotype of the Type I1 form for the stone-forming patient J, rather than homozygosity or compound heterozygosity. Several carriers in the K-L family, furthermore, displayed the same excretion patterns as patient J and her father and were also assigned the Type I1 heterozygous phenotype (Table IVA).

We predicted the genotypes of patients from the histories, pedigrees, and phenotype(s) of parents and offspring. The subtypes were clear and genotype predictions reliable when the obligate carriers of a family excreted at rates similar to those described by Rosenberg and co-workers [5]. The subtype is clearly the Type I variant, and the patients’ genotypes are homozygous, in the families of I, 0, R, S, and U, for example, since both parents of each patient excreted normal amounts of the appropriate amino acids (Table VIII). Similarly, the subtype is clearly the Type I11 variant in the G-H family, and the patients’ genotypes are homozygous. Genotypes in the K-L family, on the other hand, are heterozygous. The patients K and L have compound heterozygous genotypes for Types I1 and 111, as the healthy parents had, respectively, gross and mild cystinuria, and patient L‘s healthy offspring had gross cystinuria. We predicted compound heterozygous genotypes for E, M, and Q, patients of different families, whose parents expressed respective phenotypes of the Type I and I11 variants. Compound heterozygous genotypes were predicted tentatively for patient P, whose 9-year-old obligate carrier daughter excreted normally (Type I) and whose mother and adult brother had mild cystinuria (Type HI), and for patient A, whose adult offspring’s excretion patterns (interpreted from semi- quantitative data) were, respectively, normal (Type I) and mildly cystinuric (Type 111).

We predicted Type I1 genotypes, as mentioned earlier, in families where the obligate carrier’s arginine excretion was less in proportion to cystine and lysine than in stone- forming patients. Thus, patient J’s excretion pattern andits similarity to her father’s were the convincing evidence when we predicted a heterozygous genotype of the Type I1 form: Her excretion of cystine and the dibasic amino acids was excessive but less than that of other stone-formers. The arginine excretion, in particular, was less than that of other patients. Her healthy father’s phenotype, furthermore, resembled hers, including the

TAB

LE IV

A.

Excr

etio

n R

ates

(m

glda

y (a

)] a

nd C

once

ntra

tions

[m

g/gm

cre

atin

ine

(b)]

of

Am

ino

Aci

ds in

Fam

ilies

of S

tone

-For

min

g Pa

tient

s Who

se G

enot

ypes

Wer

e Pr

edic

ted

Hom

ozyg

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Phen

otyp

e

Fam

ily

to p

atie

nt

(yr)

a

b a

b a

b a

b a

b

Rel

atio

nshi

p A

ge

Cys

tine

Lysi

ne

Arg

inin

e O

rnith

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assi

gned

(Typ

e)

-

F Pa

tient

M

othe

r Si

ster

Si

ster

B

roth

er

I Pa

tient

Fa

ther

M

othe

r B

roth

er

0

Patie

nt

Fath

er

Mot

her

R

Patie

nt

Fath

er

Mot

her

23-3

2 54

8 45

12

22

7

15

7 10

6

22-2

5 48

6 13

13

19

901

23

621 19

5

20

681 17

16

-

-

-

-

-

-

478 8 4 4 4

428 10

12

64

694 16

10

565 11

14

1,02

3 82

9 18

11

41

22

27

14

60

43

718

588

14

11

3 3

1,55

9 1,

080

1,04

9 1,

170

30

27

12

29

1,45

0 2,

790

24

16

78

70

65 3 2 5 5 6

3 27 3 tr

1,

566

124 3 2

1,84

2 2 2

5 35 1 3 3 4

263 2 tr

1,09

0

807 3 5

1,53

0 2 1

202 2 3 2 2

130 2 tr

301

329 2 1

413 2 1

172

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1

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2

11

11

132 2

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9 I/

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I/P

367 2 1

342

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24

18

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1 55

8 42

8 66

51

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33

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49

16

13

50

40

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Mot

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49

6 8

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tr

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26

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Pa

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5 31

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1,09

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7 40

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6 Fa

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64

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61

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45

31

81

67

4

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34-3

8 56

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4 1,

799

943

927

537

458

258

22

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637

1,38

0 1,

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900

396

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73

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the

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TAB

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V B

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[m

g/da

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nd C

once

ntra

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[m

g/gm

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of

Am

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Bro

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49

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7 47

47

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51 9 35

30

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152

<17 96

1,21

2 32

9 58

9 85 7 14

17

123

42 3 22

102

422 14

74

66

0 7

174

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627 15

9

390 33

7 11

12

115 35 7 12

97

647 8 63

53

2 10

922

1,05

0 43

33

27

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3

2,50

2 84

6 10

3 52

16

16

1,74

0 1,

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260

110

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31

49

35

307

396

93

143

12

26

77

39

307

298

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1,50

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19

33

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3 1,

647

1,33

5 35

49

487

<9

<9

862 2 2

1,16

7 18 6 6 2

193 1 2 5 5

246 4 9

24 2 3

55 7

<7

<7

502 1 2

636 7 6 2 4

25 0

2 3 2 5

373 3 8

198 4

a 84

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N

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1,57

9 3 1

366 11

1

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107 2 1 4 23

186 2 15

31

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Phen

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rnith

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assi

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(Typ

e)

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a

b

96

ND

N

D

146 1 1

27 I 5 1 1

1

138 3 3 2 23

282 1 13

25

2 2

Ia

IIP

111

I1

111

111

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N

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111

111

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111

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11

11

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ther

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ster

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ster

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ster

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Pa

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(K)

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D

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ter

(L)

Son

(L)

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(L)

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12

10

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50

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18

15

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78

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B

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26

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28

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w

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iece

3

C

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ntd

34

Dau

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388

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6 12

6 41

769

208 52

8 69

3 21

5 33

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4 25

2

13

133 50

47

40

545 5

370

268 9 5

126 29

398

101 33

50

41 1

11

5 14

9 70

161 15

42

15

52

40

373 30

641

600

889

516

29

18

48

44

352

366

157

118

2,63

5 1,

078

428

213

102

64

59

344

1,37

9 81

3 74

5 42

0 56

2 21

1 35

0 17

9 21

6 15

4 4

5 40

4 13

5 79

30

17

5 16

7 12

5 14

9

490

1,22

0 14

80

39

22 4 2 12

4

473 47 4 7

658 31

24

12

15

2 18 6 3 9

944 1

33 9 3 2 12

3

336 23

3 17

393 17

11

6 11 2 6 2 2 11

97 1 5

76

75 1 1 14 4

580 14

3 2 39

8 49

55

19

46 1 15 4 6 5

292 1

68

IIC

IF

43

I1

I1

1

NN

1

N

N

I1

I1

3 I1

I1

7 I1

I1

2

111

111

14

21 1

9 11

11

I1

238 28

I1

I1

26

I1

I1 10

11

I1

33

I1

I1

1 N

N

5 I1

1

I I1

5 11

I1

I1

11

7

286 11

I

111

*Lys

ine

vari

ants

are

indi

cate

d by

sub

scri

pt “1

” ; si

blin

g ph

enot

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des

igna

ted

N(o

rmal

), in

fam

ilies

whe

re o

blig

ate

carr

iers

exp

ress

T

ype

I phe

noty

pes,

shou

ld b

e in

terp

rete

d as

eith

er N

or T

ype

I. N

D is

Not

det

erm

ined

. as

emiq

uant

itativ

e dat

a fr

om p

aper

chr

omat

ogra

phy.

bA

sym

ptom

atic

. C

Patie

nt c

onsi

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186 Kelly

TABLE V. Excretion Rates [mg/day (a)] and Concentrations [mg/gm creatinine (b)] of Amino Acids in Incomoletelv Recessive Obligate Carrier Offsoring at Different Ages

Heterozygous phenotype

Lysine Arginine Ornithine assigned Patient Carrier (yr) a b a b a b a b a b

_____ Age Cystine

- L Son 6 15 26 73 131 2 4 2 4 1111 1111

12 180 160 444 420 21 20 31 29 I1 I1 15 144 70 350 180 12 6 19 10 I1 I1

Son 10 40 39 228 227 12 12 11 11 1111 1111 15 337 149 562 271 24 11 55 26 I1 I1

Daughter 8 134 168 350 438 14 18 23 29 11 11 13 282 204 624 472 25 19 48 36 I1 11 18 215 115 745 420 31 17 49 28 I1 I1

Daughter 7 19 24 73 94 4 5 3 4 1111 1111 9 15 30 47 92 4 9 2 4 I11 111

Daughter 4 13 13 37 44 3 4 2 2 111 111 6 16 38 62 161 3 9 3 7 1111 1111

H

TABLE VI. Rates and Concentrations of Cystine and the Dibasic Amino Acids After Being Ranked With Age at Time of Sampling, Excreted by Incompletely Recessive Carrier Children of Probable Type I1 HeterozygousGenotype, Younger (-) or Older (+) Than the Median Age of 10 Years

Mean rate Mean concentration (mg/day) (mg/gm creatinine)

Amino acid - + Diff, ~ + Diff.

Cystine 54 212 158a 65 141 76b Lysine 190 466 276a 170 341 171a Arginine 8 18 loa 9 15 6 Ornithine 9 33 24a 11 23 12

~~

aSignificant a t between the 1 and 5% levels. bSignificant a t between the 5 and 10% levels.

arginine excretion. The mother’s excretion was normal, and sibs expressed normal and Type I1 heterozygous phenotypes, respectively. Genotype predictions were clearer in the other family with Type 11 carriers, as the patients K and L excreted arginine in amounts comparable to those excreted by other kinds of stone-forming patients and in far greater amounts than their Type I1 carrier relatives.

Conversely to the prediction of heterozygosity in patient J , a stone-forming patient, we predicted homozygous or compound heterozygous genotypes for two healthy relatives of stone-forming patients: patient 1’s brother, for example, who excreted at rates and


TABLE VII. Rates and Concentrations of Cystine and the Dibasic Amino Acids After Being Ranked With Age at the Time of Sampling, Excreted by Incompletely Recessive Carrier Children of Probable Type 111 Heterozygous Genotype, Younger (-) or Older (+) Than the Median Age of 5.5 Years

Mean rate Mean concentration __ (mg/day) (mg/gm creatinine)

Amino acid - + Diff. - + Diff.

Cystine 14 16 2 19 31 12 Lysine 43 60 17 54 116 62 Arginine 3 4 1 4 8 4a Ornithine 2 3 1 2 5 3a

aSignificant at the 5% level.

TABLE VIII. Prediction of Genotypes in 17 Patients With Cystinuria From Excretory Patterns of Obligate Carrier Parents and of Offspring and Other Relatives’

Phenotype Offspring Sibling Predicted

Patient Father Mother a b c a b c Other genotype

- - I/III III/?

- - I/III

A - - I 111 - -

C

F -

- - - - 111 - - -

E 1111 I - _ _ -

N N ‘lys I/I - - _ I 1111 1111

111, N - - III/III _ - _

G } 111

K } I1

H

Jb I1 N N I1 I1 II/N I I I I / P - - I /I - - -

- - _ 1111 - -

I11 I1 111 - I F IId IId II/III I1 I1 I1 L

0 I I M 111 I - - _ N N - I/III

I /I P - 1111 I - - I1 111 - I/III Q I 111 I/IIIa N - I/III

I t 1 N N - I/1

R I I1

U I I N N - I/I

_ - _ - - -

- - _ _ - _ - - -

- - _ S I I - - _

*The “lysine variant” is indicated by the subscript “1.” aAsymptomatic. bymptumatic. ‘Grandfather. dNiece and nephew.

188 Kelly

concentrations similar to hers (a Type I homozygote); and patient Q’s sister, who at age 17 also excreted in the range of stone-forming patients.

of single obligate carriers. The prediction of patient F’s homozygosity is supported, however, by the presence of similar phenotypes in the sibs and obligate carrier mother (Type I). The evidence for patient C’s genotype is weaker, since it is comprised entirely of data from one obligate carrier offspring. One might question, furthermore, the validity of assigning the phenotype in a 4-year-old child. However, we employed the more reliable coefficient, concentration, rather than rate when interpreting the excretory pattern.

in six families and in combination with the Type 111 form in five families. The Type 111 variant was the commoner of the incompletely recessive forms, appearing in one family in the homozygous state, in combination with the recessive (Type I) form in five families, in combination with the Type I1 form in one family, and in uncertain combination in one patient. Type I1 variant also appeared in a family in the heterozygous/normal state (Table 111).

The genotypes of patients F and C are less certain, as they were predicted from data

Thus, the Type I variant was present in 1 1 of 15 families - in the homozygous state

DISCUSSION

The subtyping of new cases of cystinuria, while not required for the diagnosis, is a means of expanding the taxonomy of the disease. The complexities, however, have dis- couraged all but a few investigators from studying affected families for this purpose. Our attempt was based on the premise that the refinements made in amino acid analyses since the work of Harris and associates and the delineation of excretory patterns in incompletely recessive forms by Rosenberg et a1 have simplified the task of subtyping. Thus, we were able to collect data from a large number of families and from urine analyses alone distinguish types, combinations, and subtypes. The data were numerous enough, furthermore, to consider the distribution of types. The recessive (Type I) form, for example, was the commonest in our families, all of whom lived in New York State. The distribution here, therefore, is more like that in England [4,6] and Canada [l 11 than in the homogeneous population described by Weinberger et a1 [14]. The patients with compound heterozygous disease (ie, caused by different subtypes) were about as numerous as those with homozygous disease (caused by similar subtypes), suggesting heterogeneity like that in Australian families [24].

Type I1 form, for example, is at higher risk of forming stones, because of gross cystinuria, than the Type 111 carrier. Indeed, the rare appearance of stones in successive generations is less puzzling if, apart from attributing it to homozygous-heterozygous matings [6, 111, one can identify an affected Type I1 heterozygote in the pedigree. Conversely, the truly recessive obligate carrier (Type I), eg, the daughter of patient P, is as much a carrier of the mutation as the Type I1 or Type 111 (incompletely recessive) carriers. On the other hand, the discovery that healthy siblings may excrete the amino acids in amounts suggesting biochemical homozygosity presents a challenge. Harris et a1 [4] also found anomalous “biochemical homozygotes” who did not form stones.

Type I1 heterozygous phenotype. Harris and co-workers [4] and Crawhall et a1 [17] recognized the pattern in “incompletely recessive heterozygotes” who formed stones

Knowledge of the subtype may be useful in genetic counseling. The carrier of the

The relatively low amount of arginine excreted was a discriminating feature of the


and who, in retrospect, were probably Type I1 carriers; Crawhall, Saunders, and Thompson [ 6 ] , furthermore, emphasized the value of arginine excretion in assessing heterozygosity in incompletely recessive variants, but few investigators have used the data.

The relative amount of lysine excretion was also helpful in assigning phenotypes. The fivefold ratio of lysine to cystine excreted by young obligate carriers (patient L’s sons) was, in retrospect, a prognosticator of the more expressive Type I1 phenotype they expressed when older (Table V). We predict, therefore, that another carrier in the family, patient K’s 2-year-old son, may later display the Type I1 heterozygous phenotype (Table IV).

In summary, the measurements described above enabled us to predict the genotypes of patients with cystinuria without recourse to biopsy studies or loading doses. The Type I form occurred more often than the other types and frequently in combination with the Type I11 form. Compound heterozygous forms of the disease were more common than in families previously studied. The Type I1 heterozygous phenotype was easily distinguished by a disproportionately low excretion of arginine, while the final (adult) phenotype might be prognosticated in young carriers of incompletely recessive forms by excessive lysine excretion.

ACKNOWLEDGMENTS

William Copeland, James Seeger, and Edward Leikhim carried out the amino acid analyses, Lucille Desjardins assisted with other technical aspects and Dr Margaret Hoff gave me statistical advice.

REFERENCES

1. 2.

3.

4 .

5 .

6.

7 .

8. 9.

10.

11.

12.

13.

14.

Dent CE, Rose GA: Amino acid metabolism in cystinuria. Quart J Med 20:205-219, 1951. Scriver CR, Rosenberg LE: “Amino Acid Metabolism and Its Disorders.” Philadelphia: Saunders, 1973, pp 158-161. Harris H, Warren FL: Quantitative studies o n the urinary cystine in patients with cystine stone formation and in their relatives. Ann Eugenics 18:125-171, 1953. Harris H, Mittwoch U, Robson EB, Warren FL: Phenotypes and genotypes in cystinuria. Ann Hum Genet 20:57-91,1955. Rosenberg LE, Downing S , Durant JL, Segal S : Cystinuria: Biochemical evidence for three genetically distinct diseases. J Clin Invest 45:365-371, 1966. Crawhall JC, Saunders EP, Thompson CJ: Heterozygotes for cystinuria. Ann Hum Genet 29:

Rosenberg LE, Durant JL, Albrecht I: Genetic heterogeneity in cystinuria: Evidence for allelism. Trans Assoc Am Phys 79:284-296,1966. Rosenberg LE: Cystinuria: Genetic heterogeneity and allelism. Science 154: 1341-1 343, 1966. Kelly S, Copeland W: Amino acid patterns in cystinuric families. J Med Genet 5:281-285, 1968. Kelly S, Copeland W, Leikhim E: Amino acid excretion patterns in the offspring of a “doubly heterozygous” cystine stone former. Experientia 26: 1389- 1390, 1970. Morin CL, Thompson MW, Jackson SH, Sass-Kortsak A: Biochemical and genetic studies in cystinuria: Observations on double heterozygotes of genotype 1/11. J Clin Invest 50:1961-1976, 1971. Minami R, Olek K, Wardenbach P: Cystinuric heterozygotes and cystine-loading. Humangenetik 29(2):145-149,1975. Borstrtim H, Hambraeus L: Cystinuria in Sweden. VII. Clinical, histopathological and medico- social aspects of the disease. Acta Med Scand (Suppl411) 1964. Weinberger A, Sperling 0, Rabinovitz M, Brosh S , Adam A, deVries A: Proceedings: Rarity of cystinuria Type I among cystinuric Jewsof Libyan origin. Isr J Med Sci 11:1217, 1975.

257-269,1966.

190 Kelly

15. Hambraeus L: Cystinuria in Sweden. X. Quantitative studies of the urinary amino acid excretion in cystinurics. Acta Soc Med Upsalien 69:l-22, 1964.

16. Frimpter GW: Cystinuria: Metabolism of the disulfide of cysteine and homocysteine. J Clin Invest 42:1956-1964, 1963.

17. Crawhall JC, Purkiss P, Watts RWE, Young EP: The excretion of amino acids by cystinuric patients and their relatives. Ann Hum Genet 33:149-169, 1969.

18. Crawhall JC, Scowen EF, Thompson CJ, Watts RWE: The renal clearance of amino acids in cystinuria. J Clin Invest 46:1162-1171, 1967.

19. Brand E, Harris MM, Biloon S: The excretion of a cystine complex which decomposes in the urine with the liberation of free cystine. J Biol Chem 86:315-331, 1930.

20. Efron ML, Young D, Moser HW, MacCready RA: A simple chromatographic screening test for the detection of disorders of amino acid metabolism. A technic using whole blood or urine collected on filter paper. N Engl J Med 270:1378-1383,1964.

21. “Technicon Auto Analyzer Methodology: Method File N116.” Chauncy, New York: Technicon Instruments Corporation, 1965.

22. Stein WH: A chromatographic investigation of the amino acid constituents of normal urine. J BiolChem 201:45-58, 1953.

23. Kennedy C, Shih VE, Rowland LP: Homocystinuria: A report in two siblings. Pediatrics 36: 736-741, 1965.

24. Turner B, Smith A: The New South Wales register of cystinurics: First report January to December, 1971. Med J Australia 2:1005-1007, 1973.

Edited by Charles E. Scriver

Cystinuria genotypes predicted from excretion patterns

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