Am. J. Hum. Genet. 49:804-810, 1991
Lowe Oculocerebrorenal Syndrome in a Female with a Balanced X;20 Translocation: Mapping of the X Chromosome Breakpoint 0. T. Mueller,* J. K. Hartsfield, Jr.,t L. A. Gallardo,* Y.-P. Essig,* K. L. Miller,* P. R. Papenhausen, * and T. A. Tedesco * *Pediatric Laboratories and tRegional Genetics Program, Department of Pediatrics, University of South Florida College of Medicine, Tampa
Summary A Hispanic girl with Lowe oculocerebrorenal syndrome (OCRL), an X-linked recessive condition characterized by cataracts, glaucoma, mental retardation, and proteinuria, is reported. A balanced X;20 chromosomal translocation with the X chromosome breakpoint at q26.1 was found with high-resolution trypsin-Giemsa banding. Somatic cell hybridization was used to separate the X chromosome derivative and the chromosome 20 derivative in order to position, with respect to the translocation breakpoint, several DNA loci that are linked to the Lowe syndrome locus (Xq24-q26). DXS10 and DXS53 were found to be distal to the breakpoint, whereas DXS37 and DXS42 were located proximal to it. These studies suggest that the OCRL locus lies in the region between these probes. The translocation chromosome originated from an unaffected male without a visible translocation, indicating that the most likely cause of OCRL in this patient is the de novo translocation that disrupted the OCRL locus.
Introduction
X-linked recessive disorders occurring in female patients are often associated with X; autosome translocations. The position of the X chromosome breakpoint is likely to define the position of the gene(s) causing the disorder. Lowe oculocerebrorenal syndrome (OCRL; McKusick 30900 [McKusick 1989]) occurs predominantly in males, although several affected females have been reported (Scholten 1960; Svorc et al. 1967; Harris et al. 1970; Sagel et al. 1970; Cyvin et al. 1973; Yamashina et al. 1983; Hodgson et al. 1986). Several of these patients' diagnoses or cytogenetic findings may be questionable (table 1). The lack of cataracts, as well as the similarly affected female sibling in the report of Scholten (1960), would be unusual in OCRL. The "46,XX" karyotypes in the patients of Svorc et al. (1967), Sagel et al. (1970), and Cyvin et Received May 15, 1990; final revision received June 4, 1991. Address for correspondence and reprints: 0. Thomas Mueller, Ph.D., Department of Pediatrics, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4904-0013$02.00
804
al. (1973) were not shown in these reports and were presumably not banded. Thus they may not have been able to detect a translocation such as was found by Hodgson et al. (1986) or by the present study. Linkage analysis places the OCRL locus in the region Xq24-q26 (Silver et al. 1987a; Reilly et al. 1988, 1990; Wadelius et al. 1989). These investigators identified several loci which flank the Lowe syndrome locus at close proximity. Among these loci, DXS42, DXS37, and DXS100 are very closely linked markers (Silver et al. 1987b; Wadelius et al. 1989; Reilly et al. 1990) that were proximal to the X;3 translocation breakpoint in the female OCRL patient described by Hodgson et al. (1986). Several other closely linked loci-HPRT, DXS10, DXS53, and DXS86-were distal to the breakpoint in this patient, suggesting that the X breakpoint occurs within the OCRL locus (Reilly et al. 1988b, 1990). We report the occurrence of OCRL in a young woman with an apparently balanced translocation: 46,XXt(X;20) (q26.1;qll.2) (Mueller et al. 1989). The X chromosome breakpoint, q26.1, may be different from that of the patient described by Hodgson et al. (1986), who reported it as q25. To resolve this possible discrepancy, somatic cell hybrids were
Oculocerebrorenal Syndrome in a Female
80S
Table I Females Reported with Lowe Syndrome
STATUS
Scholten (1960) Studya
FEATURE
Developmental delay Hypotonia ................ Hyporeflexia ............... Nystagmus ................
?
+ +
+ + + +
+ +
Kidney biopsy ........
-
Abnormalities of proximal tubules
-?
Hodgson et al. (1986) Study
Present Study
+
+ + + + +
+ + + + +
+ +
+ +
+
46,XX,t(14;17)
46,XX,t(X;20)
(q24;q23),t(X;3) (q25;q27)
(q26.1;q11.2)
?
+
................
Karyotype .................
IN
Cyvin et al. (1973) Study
+ +
+ Cataracts Rickets .................?.Proteinuria ................ + + Aminoaciduria ............. +
PATIENT(S)
Sagel et al. (1970) Studyb
....
+ +
OF
Svorc et al. (1967) Study
+ +
ala, lys, tyr thr, gly 46,XX
+
46,XX
46,XX
LM normal, Postmortem with EM abnormal dilated tubules and
including mitochondria
thickening of Bowman capsule
+C
LM normal, EM
nonspecific changes
NOTE. -Yamashina et al. (1983) described 21 patients with both Lowe syndrome and increased nucleotide pyrophosphatase activities. One male had no aminoaciduria. Two females were reported with elaboration of clinical features. a Details were given on female who died at 2 mo of age. A female sibling died at 15 mo with similar features. b Apparently the same patient as reported by Harris et al. (1970). c Proteinuria was markedly (10-fold) elevated. Alanine was significantly elevated, and there were mild elevations of glycine, aspartate, threonine, and 3-aminoisobutyric acid, in an otherwise normal pattern.
constructed to determine whether the breakpoint in our patient was also within the interval defined by the OCRL-linked probes. We also used polymorphic probes to establish the parental origin of the de novo translocation and investigated whether there was nonrandom X chromosome inactivation in our patient. Case Report A 14-year-old Hispanic girl presented with a history
of developmental delay, hypotonia, hyporeflexia, nystagmus, congenital cataracts, proteinuria, intermittent hematuria, slight aminoaciduria, persistent hyperchloremic metabolic acidosis, and short stature (Hartsfield et al. 1989). Her height was 128.27 cm (i.e., within 5 standard deviates), her weight was 30.4 kg (i.e., within 3 standard deviates), and her head circumference was 51.7 cm (1Oth percentile). Birth was by spontaneous vertex delivery after a term gestation, and birth weight was 3.57 kg (65th percentile). She started walking alone and said her first words at approximately 3.5 years of age. While she is bilingual (Spanish and English), her language skills and behav-
ior were immature. She reportedly tests in the trainable range of mental retardation. There were no radiologic findings of rickets, although there was evidence of healed Legg-Perthes disease of the left hip. The family history was negative for OCRL or eye abnormalities. A kidney biopsy revealed 20% of her glomeruli to have global sclerosis, with a few tubules showing hypertrophic changes. Immunofluorescence studies were negative. Electron microscopy revealed segmental obsolescence with glomerular basementmembrane attenuation and wrinkling. Occasional replacement of foot processes were noted in relation to the sclerotic loops. There was marked edema of the epithelial cells in these areas. Previously described ultrastructural features of OCRL syndrome, such as enlargement of proximal tubular mitochondria, displacement of cisternae, and prominent matrical bodies, were not noted. When she was 6 years of age, a urine screen for inborn errors of metabolism indicated that "amino acids may be very slightly elevated." Screening of current urine amino acids found a mild elevation in alanine, glycine, aspartic acid, threonine, citrulline, and beta-amino isobutyric acid, in an other-
806
wise normal pattern as determined by high-performance liquid chromatography and ninhydrin detection. Review of the literature found a small number of cases in which a female had been reported with OCRL syndrome (table 1). Three of these cases were reported to have a normal female karyotype, while one had a translocation involving the X chromosome. Our patient is noteworthy in her lack of a marked generalized aminoaciduria. While Yamashina et al. (1983), in their study of OCRL, included one male patient who had increased nucleotide pyrophosphatase activities without aminoaciduria, the presence of a generalized aminoaciduria is considered to be an integral part of the diagnosis. While we do not know why our patient has only a mild, selective aminoaciduria with nonspecific kidney changes on biopsy, she appears to have OCRL. Material and Methods High-resolution chromosome analysis was performed on the proposita, her mother, and designated somatic cell hybrids, by the method of Yunis et al. (1981), on phytohemagglutinin-stimulated peripheral lymphocytes. Somatic cell hybrids were prepared by fusing a skin fibroblast culture, derived from the patient, and mouse RAG cells which are hypoxanthine phosphoribosyl transferase (HPRT) deficient (CCL 142; American Type Culture Collection). Fusion was performed by using 42% (w/v) polyethylene glycol 1000 and 7% dimethylsulfoxide in Eagle's growth medium. Hybrid clones were cultured in the presence of HAT growth medium (10 mmol hypoxanthine/liter, 1.6 mmol thymidine/liter, 4 x 10-s mol aminopterin [in Eagle's salt solution]/liter) to select for clones retaining the human chromosome 20 translocation derivative containing Xq26-qter and, thus, the active HPRT gene. Several clones were counterselected in the presence of 5 x 10-5 mol 6-thioguanine/liter, a purine analogue that inhibits growth of HPRT-active clones, to allow isolation of clones without the chromosome 20 derivative. DNA extractions were done from white blood cells and from somatic cell hybrid cultures by using 1 mg proteinase K (Boehringer-Mannheim)/ml in 5% sodium lauryl sulfate, 10 mmol Tris pH 8.0/liter, 10 mmol NaCl/liter, and 2 mmol EDTA/liter. DNA was extracted twice in phenol and once in chloroform: isoamyl alcohol (24:1 [v/v]) and was precipitated in 0.3 M sodium acetate and isopropanol. Restriction-
Mueller et al. endonuclease digestion and electrophoresis on 1 % agarose gels were done by using standard techniques. DNA probe labeling was done by using the random oligonucleotide priming reaction of Feinberg and Vogelstein (1983). Hybridizations were performed using Nytran membranes (Schleicher & Schuell) according to the manufacturer's instructions, with a final wash using 0.1 x SSC (1 x SSC = 0.15 M sodium chloride, 0.15 M sodium citrate, pH 7.0) at 650C for 2 h. The X chromosome content of somatic cell hybrid clones was determined cytogenetically and by scoring for polymorphic DNA probes for which the patient is heterozygous. Factor IX at Xq27-q28 was supplied by J. Mandel, and pERT87-30 at Xp21-p22 was obtained from the American Type Culture Collection (ATCC), as a contribution by L. Kunkel). Probes shown to map to the Xq24-q26 region containing the OCRL locus were obtained as follows: probe p36B-2 at locus DXS10 was contributed to the ATCC by R. Nussbaum, and p43-15 at the DXS42 locus was obtained from L. Kunkel through the ATCC; probe p3ORIb at the DXS37 locus was obtained from B. White, and probe Stl6 at the DXS53 locus was donated by J. Mandel. Karyotypes of hybrid clones LSR-10 and LSR-35 were analyzed for the presence of intact X chromosomes and chromosome derivative and the chromosome 20 derivative. LSR-10 has the chromosome 20 derivative in 36/40 metaphases, including eight that have two copies. None of these metaphases contained either the intact X chromosome or the X chromosome derivative. The LSR-35 hybrid contained the X chromosome derivative (4/10 metaphases) but not the intact X chromosome (0/10) or the chromosome 20 derivative (0/10). Results
The presentation of OCRL in a female patient prompted a peripheral lymphocyte chromosome investigation revealing an apparently balanced translocation between the X chromosome and chromosome 20-46,X,t(X;20) (q26.1; qll.2)-in all 25 metaphases examined. Karyotypes of both parents were normal, indicating a de novo translocation. The position of the X chromosome breakpoint, q26.1, may be different from that described as being at Xq25 in the patient of Hodgson et al. (1986). The location of the translocation breakpoint within the G-positive Xq25 band was ruled out in the reported patient, on the basis of the absence of that dark-staining region in the
Fig. 1 . High-resolution karyotype of proband showing translocation breakpoints. Figure I High-resolution karyotype of proposita's chromosome 20 and X chromosome, with arrows indicating translocation breakpoints: 46,X,t(X,20) (q26.1; qll.2).
Mueller et al.
808
chromosome 20 derivative, following high-resolution banding (fig. 1). Both Xq25 and Xq26.1 are within the X chromosome map position of OCRL, as determined by linkage analysis. To further characterize the X chromosome translocation breakpoint with respect to the OCRL-linked probes, we constructed somatic cell hybrids by using skin fibroblasts derived from the patient and HPRTdeficient mouse RAG cells. We identified on the long and short arms of the X chromosome two polymorphic DNA probes for which the patient was heterozygous and that served to distinguish the intact X chromosome and the X chromosome derivative. One is located distal to the breakpoint: the factor IX/ TaqI RFLP (1.8 and 1.3 kb), located at band q28. The 1.3-kb fragment was determined to be paternal in origin, on the basis of the presence of homozygous 1.8 alleles in the proposita's mother (table 2). Another marker, pERT87-30/BglII (8.0 and 30 kb), identified the two X chromosome short arms. The 8.0-kb fragment was paternally derived, on the basis of homozygous 30-kb alleles in the mother. Fifty hybrid clones were selected in HAT growth medium containing an active HPRT gene which maps to Xq26. Twenty-five ofthese were further counterselected, in 6-thioguanine (GTG), for clones that lack either an intact active X chromosome or an X chromosome derivative with an active HPRT gene. All 25 HAT-selected hybrid clones
found to contain the 1.3-kb factor IX/ TaqI fragwhereas in all 6TG-counterselected hybrid clones this fragment was consistently absent (table 2), identifying it as a marker for the active X chromosome. This marker was paternally derived and was not concordant with the presence of the paternal Xp marker, the 8.0-kb pERT87-30/BglIl fragment, indicating that these markers identify the translocation chromosomes. There was also perfect concordance with the karyotype presence of both the normal X chromosome and the 30-kb pERT87-30/BglII and 1.8-kb factor IX/ TaqI fragments. These data indicate that there was nonrandom lyonization in our patient and identify the normal, maternally derived X chromosome as that which is preferentially inactivated. One HAT-selected hybrid clone, LSR-10, was identified that contained the 1.3-kb factor IX/ TaqI fragment but neither of the pERT87-30/BglII markers, indicating that it contained only the chromosome 20 derivative and not the intact X chromosome or the X chromosome derivative (table 2). One of the 6TGcounterselected hybrid clones, LSR-35, contained the 8.0-kb pERT87-30/BglII fragment but neither of the factor IX/ TaqI markers, indicating that it had only the X chromosome derivative. When these hybrid clones were tested for the presence of the OCRLlinked markers, the DXS10 and DXS53 probes identified homologous sequences in the LSR-10 clone (fig. were
ment,
Table 2 Presence of OCRL-Linked Loci in Somatic Cell Hybrids
Xp MARKER DXS164
DSX10 p36B-2
DXS42 p43-15
p30RIb
DXS53 Stl6
BglII
F9 Factor IX TaqI
TaqI
BglII
PvuII
BglII
30/30 8.0/30
1.8/1.8 1.8/1.3
7.0/5.0 7.0/7.0
10.0/6.0 10.0/6.0
7.5/7.5 7.5/7.5
9.4/9.4 9.4/9.4
...
...
...
8.0 8.0 ... ... 8.0
1.3(w) 1.3 1.8/1.3 1.3 1.3
7.0 7.0 7.0 7.0 7.0
10.0 10.0 6.0/10.0 ... ND
ND ND ND ... ND
ND ND ND 9.4 ND
30/8.0 8.0 30/8.0 30
1.8 ... 1.8 1.8
7.0 ... 7.0 7.0
6.0/10.0 10.0 ND 6.0
ND 7.5 ND ND
ND
pERT87-30 KARYOTYPE
Mother of proband 46,XX Proband ................ 46,X t(X;20) M ouse RAG cells ......... HAT-selected hybrids: X der, 20 der LSR-1 ............... X der, 20 der LSR-7 ............... LSR-8 ............... X, X der, 20 der 20 der LSR-10 ............... X der, 20 der LSR-16 ............... 6TG-selected hybrids: LSR-27 ............... X, X der X der LSR-35 ............... LSR-37 ............... X, X der X LSR-42 ............... .......
OCRL-LINKED PROBES
Xq MARKER
NOTE.-An ellipsis indicates that no bands were visualized. ND = not determined.
DXS37
....
...
ND ND
809
Oculocerebrorenal Syndrome in a Female 2). This indicated that they map distal to the Xq26.1 breakpoint. The DXS42 and DXS37 probes hybridized only with the LSR-35 hybrid clone (fig. 2), indicating that they map proximal to the Xq26.1 breakpoint.
Discussion
The OCRL phenotype in female patients can be explained as resulting from either (a) the rare occurrence of simultaneous deleterious mutations on both X chromosomes, (b) disproportionate inactivation of the normal X chromosome in appropriate tissues, (c) the occurrence of an X;autosomal translocation along with a coincidental mutation within the OCRL locus, or (d) the occurrence of a translocation within the
LSR LSR
LSR LSR 10 35
10
30 -
1.8
-
1.3
-
8DXS 1 64/Bgl 11
Factor IX/Taq 7.0
5.0
-
3.8 3.3
-
-
-
DXS 1O/Taq I
9.4
35
DXS42/Bgl If
7.5
-
DXS53/Bgl I
-
DXS37/Pvu I
Southern blot analysis of proposita, her mother, Figure 2 and somatic cell hybrid clones LSR-10 and LSR-35 derived from skin fibroblasts of proposita. The following DNA loci were visualized: DXS164 (pERT87-30/BglII) as the Xp marker, factor IX/ TaqI as the Xq marker, and the OCRL-Iinked probes DXS10, DXS37, DXS42, and DXS53. The DXS42 blot illustrates the constant bands at this locus; the polymorphic bands were not adequately visualized. According to karyotyping, the somatic cell hybrid LSR-10 contained the chromosome 20 derivative and none of the X chromosome derivative or intact X chromosomes. The hybrid LSR-35 contained the X chromosome derivative and none of either the chromosome 20 derivative or the intact X chromosome.
OCRL locus. The latter two possibilities would involve a preferential inactivation of the normal X chromosome, to prevent functional monosomy of the autosomal segment involved. The data presented here indicate that Iyonization was not random in the described female OCRL patient. Somatic cell hybrids selected for an active HPRT gene consistently retained one X-chromosomal derivative, as defined by the 1.3kb factor IX/ TaqI fragment, whereas no hybrids counterselected in 6TG had this marker. The observation that neither of the pERT87-30/BglII alleles was consistently observed in the HAT-selected hybrid clones further indicates that the normal X chromosome was the inactivated chromosome. This is consistent with reported observations of X;autosomal translocations that show preferential inactivation of the normal X chromosome. The alleles that appeared in the X chromosome derivative and in the chromosome 20 derivative originated from a unaffected male without a visible chromosome rearrangement. This suggests that the OCRL mutation and the X;20 translocation most likely occurred concurrently and that the translocation disrupts the function of the OCRL locus in this patient. Our OCRL patient has an apparently balanced de novo X;20 translocation with the X chromosome breakpoint at q26.1 This is in close proximity to the map position of the OCRL locus, as determined by linkage studies (Silver et al. 1987a, 1987b; Reilly et al. 1988a, 1990; Wadelius et al. 1989) and a reported X;autosomal translocation (Hodgson et al. 1986). The X chromosome breakpoints, in the patient described by Hodgson et al. (1986) and our patient, may not be significantly different (Xq2S vs. Xq26. 1), since the karyotype of the Hodgson et al. patient was not done by using high-resolution banding. Our findings indicate that the translocation in our patient is within the X-chromosomal region defined by the DNA interval between the DXS42/DXS37 and DXS10/DXS53 loci. This is the same interval interrupted by the translocation in the Hodgson et al. patient (Reilly et al. 198 8a, 1989, 1990), suggesting that both breakpoints may be within the OCRL locus. Somatic cell hybrids containing the derivative chromosomes from two different translocations within the OCRL locus should allow the facile identification of candidate genes.
Acknowledgments We would like to thank Dr. Alphonso Campos for the information on the patient's renal status, Dr. Boris Kousseff
810 for his suggestions on the manuscript, Dr. J. Mandel for his contribution of the factor IX and Stl6 probes, and Dr. B. White for the p3ORIb probe. This study was supported in part by Physician Scientist Award DE00243 (to J.K.H.) from the National Institute for Dental Research.
References Cyvin KB, Weudemann H, Bathen J (1973) Lowes syndrome. Acta Paediatr Scand 62:309-312 Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Harris LS, Gitter KA, Galin A, Plechaty GP (1970) Oculocerebro-renal syndrome: report of a case in a baby girl. Br J Ophthalmol 54:278-280 Hartsfield JK Jr, Papenhausen PR, Kousseff BG (1989) Features of Lowe syndrome in a female with a translocation involving the X chromosome. Clin Res 37:52A Hodgson SV, HeckmattJZ, Hughes E, CrollaJA, Dubowitz V, Bobrow M (1986) A balanced de novo X/autosomal translocation in a girl with manifestations of Lowe syndrome. Am J Med Genet 23:837-847 McKusick VA (1989) Lowe oculocerebrorenal syndrome. In: Mendelian inheritance in man, 8th ed. Johns Hopkins University Press, Baltimore, pp 1335-1336 Mueller OT, Hartsfield JK Jr, Papenhausen PR (1989) An X/autosomal translocation in a woman with some features of Lowe syndrome. Paper presented at the March of Dimes-Birth Defects Conference, Boston, July 10-12 Reilly DS, Lewis RA, Ledbetter DH, Nussbaum RL (198 8a) Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment. Am J Hum Genet 42:748-755 Reilly DS, Lewis RA, Nussbaum RL (198 8b) FIGE mapping
Mueller et al. of the Xq25 breakpoint in a female with Lowe syndrome and an X;3 translocation. Am J Hum Genet 43 [Suppl]: A94 (1989) Genetic and physical mapping of Xq24-q25 markers flanking Lowe syndrome. Am J Hum Genet 45 [Suppl]: A158 (1990) Genetic and physical mapping of Xq24-q26 markers flanking the Lowe oculocerebrorenal syndrome. Genomics 8:62-70 Sagel I, Ores RO, Yuceoglu AM (1970) Renal function and morphology in a girl with oculocerebrorenal syndrome. J Pediatr 77:124-127 Scholten HG (1960) Een meisje met het syndroom van Lowe. Maandschr Kindergeneeskd 28:251-255 Silver DN, Lewis RA, Ledbetter DH, Bobrow M, Nussbaum RL (1987a) Identification of tightly-linked flanking markers for Lowe syndrome. Cytogenet Cell Genet 46:692 Silver DN, Lewis RA, Nussbaum RL (1987b) Mapping the Lowe oculocerebrorenal syndrome to Xq24-q26 by use of restriction fragment length polymorphisms. J Clin Invest 79:282-285 Svorc J, Masopust J, Komarkova A, Macek M, Hyanek J (1967) Oculocerebrorenal syndrome in a female child. Am J Dis Child 114:186-190 Wadelius C, Fagerholm P, Pettersson U, Anneren G (1989) Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24-q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis. Am J Hum Genet 44:241-247 Yamashina I, Yoshida H, Fukui S, Funakoshi 1 (1983) Biochemical studies on Lowe's syndrome. Mol Cell Biochem 52:107-124 YuniaJJ, Bloomfield CD, Ensrud K (1981) All patients with acute nonlymphocytic leukemia may have a chromosomal defect. N Engl J Med 305:135-139
Lowe Oculocerebrorenal Syndrome in a Female with a Balanced X;20 Translocation: Mapping of the X Chromosome Breakpoint 0. T. Mueller,* J. K. Hartsfield, Jr.,t L. A. Gallardo,* Y.-P. Essig,* K. L. Miller,* P. R. Papenhausen, * and T. A. Tedesco * *Pediatric Laboratories and tRegional Genetics Program, Department of Pediatrics, University of South Florida College of Medicine, Tampa
Summary A Hispanic girl with Lowe oculocerebrorenal syndrome (OCRL), an X-linked recessive condition characterized by cataracts, glaucoma, mental retardation, and proteinuria, is reported. A balanced X;20 chromosomal translocation with the X chromosome breakpoint at q26.1 was found with high-resolution trypsin-Giemsa banding. Somatic cell hybridization was used to separate the X chromosome derivative and the chromosome 20 derivative in order to position, with respect to the translocation breakpoint, several DNA loci that are linked to the Lowe syndrome locus (Xq24-q26). DXS10 and DXS53 were found to be distal to the breakpoint, whereas DXS37 and DXS42 were located proximal to it. These studies suggest that the OCRL locus lies in the region between these probes. The translocation chromosome originated from an unaffected male without a visible translocation, indicating that the most likely cause of OCRL in this patient is the de novo translocation that disrupted the OCRL locus.
Introduction
X-linked recessive disorders occurring in female patients are often associated with X; autosome translocations. The position of the X chromosome breakpoint is likely to define the position of the gene(s) causing the disorder. Lowe oculocerebrorenal syndrome (OCRL; McKusick 30900 [McKusick 1989]) occurs predominantly in males, although several affected females have been reported (Scholten 1960; Svorc et al. 1967; Harris et al. 1970; Sagel et al. 1970; Cyvin et al. 1973; Yamashina et al. 1983; Hodgson et al. 1986). Several of these patients' diagnoses or cytogenetic findings may be questionable (table 1). The lack of cataracts, as well as the similarly affected female sibling in the report of Scholten (1960), would be unusual in OCRL. The "46,XX" karyotypes in the patients of Svorc et al. (1967), Sagel et al. (1970), and Cyvin et Received May 15, 1990; final revision received June 4, 1991. Address for correspondence and reprints: 0. Thomas Mueller, Ph.D., Department of Pediatrics, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612. i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4904-0013$02.00
804
al. (1973) were not shown in these reports and were presumably not banded. Thus they may not have been able to detect a translocation such as was found by Hodgson et al. (1986) or by the present study. Linkage analysis places the OCRL locus in the region Xq24-q26 (Silver et al. 1987a; Reilly et al. 1988, 1990; Wadelius et al. 1989). These investigators identified several loci which flank the Lowe syndrome locus at close proximity. Among these loci, DXS42, DXS37, and DXS100 are very closely linked markers (Silver et al. 1987b; Wadelius et al. 1989; Reilly et al. 1990) that were proximal to the X;3 translocation breakpoint in the female OCRL patient described by Hodgson et al. (1986). Several other closely linked loci-HPRT, DXS10, DXS53, and DXS86-were distal to the breakpoint in this patient, suggesting that the X breakpoint occurs within the OCRL locus (Reilly et al. 1988b, 1990). We report the occurrence of OCRL in a young woman with an apparently balanced translocation: 46,XXt(X;20) (q26.1;qll.2) (Mueller et al. 1989). The X chromosome breakpoint, q26.1, may be different from that of the patient described by Hodgson et al. (1986), who reported it as q25. To resolve this possible discrepancy, somatic cell hybrids were
Oculocerebrorenal Syndrome in a Female
80S
Table I Females Reported with Lowe Syndrome
STATUS
Scholten (1960) Studya
FEATURE
Developmental delay Hypotonia ................ Hyporeflexia ............... Nystagmus ................
?
+ +
+ + + +
+ +
Kidney biopsy ........
-
Abnormalities of proximal tubules
-?
Hodgson et al. (1986) Study
Present Study
+
+ + + + +
+ + + + +
+ +
+ +
+
46,XX,t(14;17)
46,XX,t(X;20)
(q24;q23),t(X;3) (q25;q27)
(q26.1;q11.2)
?
+
................
Karyotype .................
IN
Cyvin et al. (1973) Study
+ +
+ Cataracts Rickets .................?.Proteinuria ................ + + Aminoaciduria ............. +
PATIENT(S)
Sagel et al. (1970) Studyb
....
+ +
OF
Svorc et al. (1967) Study
+ +
ala, lys, tyr thr, gly 46,XX
+
46,XX
46,XX
LM normal, Postmortem with EM abnormal dilated tubules and
including mitochondria
thickening of Bowman capsule
+C
LM normal, EM
nonspecific changes
NOTE. -Yamashina et al. (1983) described 21 patients with both Lowe syndrome and increased nucleotide pyrophosphatase activities. One male had no aminoaciduria. Two females were reported with elaboration of clinical features. a Details were given on female who died at 2 mo of age. A female sibling died at 15 mo with similar features. b Apparently the same patient as reported by Harris et al. (1970). c Proteinuria was markedly (10-fold) elevated. Alanine was significantly elevated, and there were mild elevations of glycine, aspartate, threonine, and 3-aminoisobutyric acid, in an otherwise normal pattern.
constructed to determine whether the breakpoint in our patient was also within the interval defined by the OCRL-linked probes. We also used polymorphic probes to establish the parental origin of the de novo translocation and investigated whether there was nonrandom X chromosome inactivation in our patient. Case Report A 14-year-old Hispanic girl presented with a history
of developmental delay, hypotonia, hyporeflexia, nystagmus, congenital cataracts, proteinuria, intermittent hematuria, slight aminoaciduria, persistent hyperchloremic metabolic acidosis, and short stature (Hartsfield et al. 1989). Her height was 128.27 cm (i.e., within 5 standard deviates), her weight was 30.4 kg (i.e., within 3 standard deviates), and her head circumference was 51.7 cm (1Oth percentile). Birth was by spontaneous vertex delivery after a term gestation, and birth weight was 3.57 kg (65th percentile). She started walking alone and said her first words at approximately 3.5 years of age. While she is bilingual (Spanish and English), her language skills and behav-
ior were immature. She reportedly tests in the trainable range of mental retardation. There were no radiologic findings of rickets, although there was evidence of healed Legg-Perthes disease of the left hip. The family history was negative for OCRL or eye abnormalities. A kidney biopsy revealed 20% of her glomeruli to have global sclerosis, with a few tubules showing hypertrophic changes. Immunofluorescence studies were negative. Electron microscopy revealed segmental obsolescence with glomerular basementmembrane attenuation and wrinkling. Occasional replacement of foot processes were noted in relation to the sclerotic loops. There was marked edema of the epithelial cells in these areas. Previously described ultrastructural features of OCRL syndrome, such as enlargement of proximal tubular mitochondria, displacement of cisternae, and prominent matrical bodies, were not noted. When she was 6 years of age, a urine screen for inborn errors of metabolism indicated that "amino acids may be very slightly elevated." Screening of current urine amino acids found a mild elevation in alanine, glycine, aspartic acid, threonine, citrulline, and beta-amino isobutyric acid, in an other-
806
wise normal pattern as determined by high-performance liquid chromatography and ninhydrin detection. Review of the literature found a small number of cases in which a female had been reported with OCRL syndrome (table 1). Three of these cases were reported to have a normal female karyotype, while one had a translocation involving the X chromosome. Our patient is noteworthy in her lack of a marked generalized aminoaciduria. While Yamashina et al. (1983), in their study of OCRL, included one male patient who had increased nucleotide pyrophosphatase activities without aminoaciduria, the presence of a generalized aminoaciduria is considered to be an integral part of the diagnosis. While we do not know why our patient has only a mild, selective aminoaciduria with nonspecific kidney changes on biopsy, she appears to have OCRL. Material and Methods High-resolution chromosome analysis was performed on the proposita, her mother, and designated somatic cell hybrids, by the method of Yunis et al. (1981), on phytohemagglutinin-stimulated peripheral lymphocytes. Somatic cell hybrids were prepared by fusing a skin fibroblast culture, derived from the patient, and mouse RAG cells which are hypoxanthine phosphoribosyl transferase (HPRT) deficient (CCL 142; American Type Culture Collection). Fusion was performed by using 42% (w/v) polyethylene glycol 1000 and 7% dimethylsulfoxide in Eagle's growth medium. Hybrid clones were cultured in the presence of HAT growth medium (10 mmol hypoxanthine/liter, 1.6 mmol thymidine/liter, 4 x 10-s mol aminopterin [in Eagle's salt solution]/liter) to select for clones retaining the human chromosome 20 translocation derivative containing Xq26-qter and, thus, the active HPRT gene. Several clones were counterselected in the presence of 5 x 10-5 mol 6-thioguanine/liter, a purine analogue that inhibits growth of HPRT-active clones, to allow isolation of clones without the chromosome 20 derivative. DNA extractions were done from white blood cells and from somatic cell hybrid cultures by using 1 mg proteinase K (Boehringer-Mannheim)/ml in 5% sodium lauryl sulfate, 10 mmol Tris pH 8.0/liter, 10 mmol NaCl/liter, and 2 mmol EDTA/liter. DNA was extracted twice in phenol and once in chloroform: isoamyl alcohol (24:1 [v/v]) and was precipitated in 0.3 M sodium acetate and isopropanol. Restriction-
Mueller et al. endonuclease digestion and electrophoresis on 1 % agarose gels were done by using standard techniques. DNA probe labeling was done by using the random oligonucleotide priming reaction of Feinberg and Vogelstein (1983). Hybridizations were performed using Nytran membranes (Schleicher & Schuell) according to the manufacturer's instructions, with a final wash using 0.1 x SSC (1 x SSC = 0.15 M sodium chloride, 0.15 M sodium citrate, pH 7.0) at 650C for 2 h. The X chromosome content of somatic cell hybrid clones was determined cytogenetically and by scoring for polymorphic DNA probes for which the patient is heterozygous. Factor IX at Xq27-q28 was supplied by J. Mandel, and pERT87-30 at Xp21-p22 was obtained from the American Type Culture Collection (ATCC), as a contribution by L. Kunkel). Probes shown to map to the Xq24-q26 region containing the OCRL locus were obtained as follows: probe p36B-2 at locus DXS10 was contributed to the ATCC by R. Nussbaum, and p43-15 at the DXS42 locus was obtained from L. Kunkel through the ATCC; probe p3ORIb at the DXS37 locus was obtained from B. White, and probe Stl6 at the DXS53 locus was donated by J. Mandel. Karyotypes of hybrid clones LSR-10 and LSR-35 were analyzed for the presence of intact X chromosomes and chromosome derivative and the chromosome 20 derivative. LSR-10 has the chromosome 20 derivative in 36/40 metaphases, including eight that have two copies. None of these metaphases contained either the intact X chromosome or the X chromosome derivative. The LSR-35 hybrid contained the X chromosome derivative (4/10 metaphases) but not the intact X chromosome (0/10) or the chromosome 20 derivative (0/10). Results
The presentation of OCRL in a female patient prompted a peripheral lymphocyte chromosome investigation revealing an apparently balanced translocation between the X chromosome and chromosome 20-46,X,t(X;20) (q26.1; qll.2)-in all 25 metaphases examined. Karyotypes of both parents were normal, indicating a de novo translocation. The position of the X chromosome breakpoint, q26.1, may be different from that described as being at Xq25 in the patient of Hodgson et al. (1986). The location of the translocation breakpoint within the G-positive Xq25 band was ruled out in the reported patient, on the basis of the absence of that dark-staining region in the
Fig. 1 . High-resolution karyotype of proband showing translocation breakpoints. Figure I High-resolution karyotype of proposita's chromosome 20 and X chromosome, with arrows indicating translocation breakpoints: 46,X,t(X,20) (q26.1; qll.2).
Mueller et al.
808
chromosome 20 derivative, following high-resolution banding (fig. 1). Both Xq25 and Xq26.1 are within the X chromosome map position of OCRL, as determined by linkage analysis. To further characterize the X chromosome translocation breakpoint with respect to the OCRL-linked probes, we constructed somatic cell hybrids by using skin fibroblasts derived from the patient and HPRTdeficient mouse RAG cells. We identified on the long and short arms of the X chromosome two polymorphic DNA probes for which the patient was heterozygous and that served to distinguish the intact X chromosome and the X chromosome derivative. One is located distal to the breakpoint: the factor IX/ TaqI RFLP (1.8 and 1.3 kb), located at band q28. The 1.3-kb fragment was determined to be paternal in origin, on the basis of the presence of homozygous 1.8 alleles in the proposita's mother (table 2). Another marker, pERT87-30/BglII (8.0 and 30 kb), identified the two X chromosome short arms. The 8.0-kb fragment was paternally derived, on the basis of homozygous 30-kb alleles in the mother. Fifty hybrid clones were selected in HAT growth medium containing an active HPRT gene which maps to Xq26. Twenty-five ofthese were further counterselected, in 6-thioguanine (GTG), for clones that lack either an intact active X chromosome or an X chromosome derivative with an active HPRT gene. All 25 HAT-selected hybrid clones
found to contain the 1.3-kb factor IX/ TaqI fragwhereas in all 6TG-counterselected hybrid clones this fragment was consistently absent (table 2), identifying it as a marker for the active X chromosome. This marker was paternally derived and was not concordant with the presence of the paternal Xp marker, the 8.0-kb pERT87-30/BglIl fragment, indicating that these markers identify the translocation chromosomes. There was also perfect concordance with the karyotype presence of both the normal X chromosome and the 30-kb pERT87-30/BglII and 1.8-kb factor IX/ TaqI fragments. These data indicate that there was nonrandom lyonization in our patient and identify the normal, maternally derived X chromosome as that which is preferentially inactivated. One HAT-selected hybrid clone, LSR-10, was identified that contained the 1.3-kb factor IX/ TaqI fragment but neither of the pERT87-30/BglII markers, indicating that it contained only the chromosome 20 derivative and not the intact X chromosome or the X chromosome derivative (table 2). One of the 6TGcounterselected hybrid clones, LSR-35, contained the 8.0-kb pERT87-30/BglII fragment but neither of the factor IX/ TaqI markers, indicating that it had only the X chromosome derivative. When these hybrid clones were tested for the presence of the OCRLlinked markers, the DXS10 and DXS53 probes identified homologous sequences in the LSR-10 clone (fig. were
ment,
Table 2 Presence of OCRL-Linked Loci in Somatic Cell Hybrids
Xp MARKER DXS164
DSX10 p36B-2
DXS42 p43-15
p30RIb
DXS53 Stl6
BglII
F9 Factor IX TaqI
TaqI
BglII
PvuII
BglII
30/30 8.0/30
1.8/1.8 1.8/1.3
7.0/5.0 7.0/7.0
10.0/6.0 10.0/6.0
7.5/7.5 7.5/7.5
9.4/9.4 9.4/9.4
...
...
...
8.0 8.0 ... ... 8.0
1.3(w) 1.3 1.8/1.3 1.3 1.3
7.0 7.0 7.0 7.0 7.0
10.0 10.0 6.0/10.0 ... ND
ND ND ND ... ND
ND ND ND 9.4 ND
30/8.0 8.0 30/8.0 30
1.8 ... 1.8 1.8
7.0 ... 7.0 7.0
6.0/10.0 10.0 ND 6.0
ND 7.5 ND ND
ND
pERT87-30 KARYOTYPE
Mother of proband 46,XX Proband ................ 46,X t(X;20) M ouse RAG cells ......... HAT-selected hybrids: X der, 20 der LSR-1 ............... X der, 20 der LSR-7 ............... LSR-8 ............... X, X der, 20 der 20 der LSR-10 ............... X der, 20 der LSR-16 ............... 6TG-selected hybrids: LSR-27 ............... X, X der X der LSR-35 ............... LSR-37 ............... X, X der X LSR-42 ............... .......
OCRL-LINKED PROBES
Xq MARKER
NOTE.-An ellipsis indicates that no bands were visualized. ND = not determined.
DXS37
....
...
ND ND
809
Oculocerebrorenal Syndrome in a Female 2). This indicated that they map distal to the Xq26.1 breakpoint. The DXS42 and DXS37 probes hybridized only with the LSR-35 hybrid clone (fig. 2), indicating that they map proximal to the Xq26.1 breakpoint.
Discussion
The OCRL phenotype in female patients can be explained as resulting from either (a) the rare occurrence of simultaneous deleterious mutations on both X chromosomes, (b) disproportionate inactivation of the normal X chromosome in appropriate tissues, (c) the occurrence of an X;autosomal translocation along with a coincidental mutation within the OCRL locus, or (d) the occurrence of a translocation within the
LSR LSR
LSR LSR 10 35
10
30 -
1.8
-
1.3
-
8DXS 1 64/Bgl 11
Factor IX/Taq 7.0
5.0
-
3.8 3.3
-
-
-
DXS 1O/Taq I
9.4
35
DXS42/Bgl If
7.5
-
DXS53/Bgl I
-
DXS37/Pvu I
Southern blot analysis of proposita, her mother, Figure 2 and somatic cell hybrid clones LSR-10 and LSR-35 derived from skin fibroblasts of proposita. The following DNA loci were visualized: DXS164 (pERT87-30/BglII) as the Xp marker, factor IX/ TaqI as the Xq marker, and the OCRL-Iinked probes DXS10, DXS37, DXS42, and DXS53. The DXS42 blot illustrates the constant bands at this locus; the polymorphic bands were not adequately visualized. According to karyotyping, the somatic cell hybrid LSR-10 contained the chromosome 20 derivative and none of the X chromosome derivative or intact X chromosomes. The hybrid LSR-35 contained the X chromosome derivative and none of either the chromosome 20 derivative or the intact X chromosome.
OCRL locus. The latter two possibilities would involve a preferential inactivation of the normal X chromosome, to prevent functional monosomy of the autosomal segment involved. The data presented here indicate that Iyonization was not random in the described female OCRL patient. Somatic cell hybrids selected for an active HPRT gene consistently retained one X-chromosomal derivative, as defined by the 1.3kb factor IX/ TaqI fragment, whereas no hybrids counterselected in 6TG had this marker. The observation that neither of the pERT87-30/BglII alleles was consistently observed in the HAT-selected hybrid clones further indicates that the normal X chromosome was the inactivated chromosome. This is consistent with reported observations of X;autosomal translocations that show preferential inactivation of the normal X chromosome. The alleles that appeared in the X chromosome derivative and in the chromosome 20 derivative originated from a unaffected male without a visible chromosome rearrangement. This suggests that the OCRL mutation and the X;20 translocation most likely occurred concurrently and that the translocation disrupts the function of the OCRL locus in this patient. Our OCRL patient has an apparently balanced de novo X;20 translocation with the X chromosome breakpoint at q26.1 This is in close proximity to the map position of the OCRL locus, as determined by linkage studies (Silver et al. 1987a, 1987b; Reilly et al. 1988a, 1990; Wadelius et al. 1989) and a reported X;autosomal translocation (Hodgson et al. 1986). The X chromosome breakpoints, in the patient described by Hodgson et al. (1986) and our patient, may not be significantly different (Xq2S vs. Xq26. 1), since the karyotype of the Hodgson et al. patient was not done by using high-resolution banding. Our findings indicate that the translocation in our patient is within the X-chromosomal region defined by the DNA interval between the DXS42/DXS37 and DXS10/DXS53 loci. This is the same interval interrupted by the translocation in the Hodgson et al. patient (Reilly et al. 198 8a, 1989, 1990), suggesting that both breakpoints may be within the OCRL locus. Somatic cell hybrids containing the derivative chromosomes from two different translocations within the OCRL locus should allow the facile identification of candidate genes.
Acknowledgments We would like to thank Dr. Alphonso Campos for the information on the patient's renal status, Dr. Boris Kousseff
810 for his suggestions on the manuscript, Dr. J. Mandel for his contribution of the factor IX and Stl6 probes, and Dr. B. White for the p3ORIb probe. This study was supported in part by Physician Scientist Award DE00243 (to J.K.H.) from the National Institute for Dental Research.
References Cyvin KB, Weudemann H, Bathen J (1973) Lowes syndrome. Acta Paediatr Scand 62:309-312 Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Harris LS, Gitter KA, Galin A, Plechaty GP (1970) Oculocerebro-renal syndrome: report of a case in a baby girl. Br J Ophthalmol 54:278-280 Hartsfield JK Jr, Papenhausen PR, Kousseff BG (1989) Features of Lowe syndrome in a female with a translocation involving the X chromosome. Clin Res 37:52A Hodgson SV, HeckmattJZ, Hughes E, CrollaJA, Dubowitz V, Bobrow M (1986) A balanced de novo X/autosomal translocation in a girl with manifestations of Lowe syndrome. Am J Med Genet 23:837-847 McKusick VA (1989) Lowe oculocerebrorenal syndrome. In: Mendelian inheritance in man, 8th ed. Johns Hopkins University Press, Baltimore, pp 1335-1336 Mueller OT, Hartsfield JK Jr, Papenhausen PR (1989) An X/autosomal translocation in a woman with some features of Lowe syndrome. Paper presented at the March of Dimes-Birth Defects Conference, Boston, July 10-12 Reilly DS, Lewis RA, Ledbetter DH, Nussbaum RL (198 8a) Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment. Am J Hum Genet 42:748-755 Reilly DS, Lewis RA, Nussbaum RL (198 8b) FIGE mapping
Mueller et al. of the Xq25 breakpoint in a female with Lowe syndrome and an X;3 translocation. Am J Hum Genet 43 [Suppl]: A94 (1989) Genetic and physical mapping of Xq24-q25 markers flanking Lowe syndrome. Am J Hum Genet 45 [Suppl]: A158 (1990) Genetic and physical mapping of Xq24-q26 markers flanking the Lowe oculocerebrorenal syndrome. Genomics 8:62-70 Sagel I, Ores RO, Yuceoglu AM (1970) Renal function and morphology in a girl with oculocerebrorenal syndrome. J Pediatr 77:124-127 Scholten HG (1960) Een meisje met het syndroom van Lowe. Maandschr Kindergeneeskd 28:251-255 Silver DN, Lewis RA, Ledbetter DH, Bobrow M, Nussbaum RL (1987a) Identification of tightly-linked flanking markers for Lowe syndrome. Cytogenet Cell Genet 46:692 Silver DN, Lewis RA, Nussbaum RL (1987b) Mapping the Lowe oculocerebrorenal syndrome to Xq24-q26 by use of restriction fragment length polymorphisms. J Clin Invest 79:282-285 Svorc J, Masopust J, Komarkova A, Macek M, Hyanek J (1967) Oculocerebrorenal syndrome in a female child. Am J Dis Child 114:186-190 Wadelius C, Fagerholm P, Pettersson U, Anneren G (1989) Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24-q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis. Am J Hum Genet 44:241-247 Yamashina I, Yoshida H, Fukui S, Funakoshi 1 (1983) Biochemical studies on Lowe's syndrome. Mol Cell Biochem 52:107-124 YuniaJJ, Bloomfield CD, Ensrud K (1981) All patients with acute nonlymphocytic leukemia may have a chromosomal defect. N Engl J Med 305:135-139
