American Journal of Medical Genetics 129A:1 –7 (2004)

Rapid Publication Undetectable Maternal Serum uE3 and Postnatal Abnormal Sterol and Steroid Metabolism in Antley–Bixler Syndrome Deborah L. Cragun,1 Sharon K. Trumpy,2 Cedric H.L. Shackleton,3 Richard I. Kelley,4,5 Nancy D. Leslie,1,6 Neil P. Mulrooney,1,6 and Robert J. Hopkin1,6* 1

Cincinnati Children’s Hospital Medical Center, Division of Human Genetics, Cincinnati, Ohio Bethesda North Perinatal Center, Cincinnati, Ohio 3 Children’s Hospital Oakland Research Institute, Oakland, California 4 Kennedy Krieger Institute, Baltimore, Maryland 5 Johns Hopkins Medical Institutions, Department of Pediatrics, Baltimore, Maryland 6 University of Cincinnati, College of Medicine, Cincinnati, Ohio 2

Antley–Bixler syndrome (ABS) is a rare condition characterized by radiohumeral synostosis, craniosynostosis, midface hypoplasia, bowing of the femora, multiple joint contractures, and urogenital defects. Several reports have implicated errors of steroid or sterol metabolism in the pathogenesis of ABS. Evidence for this has included association with maternal luteomas, fetal 21-hydroxylase deficiency, early pregnancy exposure to high-dose fluconazole, lanosterol 14a-demethylase deficiency, and a unique urinary steroid profile consistent with apparent pregnene hydroxylation deficiency (APHD). We report two sibs with classic ABS. During both pregnancies, mid-trimester maternal serum screening demonstrated undetectable levels of uncongugated estriol (uE3). The brother had ambiguous genitalia and increased serum levels of progesterone and 17-a-hydroxyprogesterone. Postnatal tests performed on the sister demonstrated both the unique urinary steroid profile that defines APHD and evidence of impaired lanosterol 14-a-demethylase activity. Our results suggest that in at least some patients with ABS, the skeletal findings and altered steroidogenesis are not associated with genes specific to individual sterol or steroid pathways but rather are related to an element, such as NADPH cytochrome P450 reductase (CPR) or cytochrome b5 (CYb5), that is common to all of these pathways. ß 2004 Wiley-Liss, Inc. KEY WORDS:

Antley–Bixler syndrome; steroidogenesis; cholesterol; 14-ademethylase; 21-hydroxylase deficiency; 17-hydroxylase deficiency; apparent pregnene hydroxylation deficiency; low estriol; genital ambiguity; cytochrome p450 reductase

*Correspondence to: Robert J. Hopkin, Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 4006, Cincinnati, OH 45229-3039. E-mail: [email protected] Received 24 January 2004; Accepted 27 February 2004 DOI 10.1002/ajmg.a.30170

ß 2004 Wiley-Liss, Inc.

INTRODUCTION Antley–Bixler syndrome (ABS) (OMIM #207410) is a rare disorder characterized by radiohumeral synostosis, craniosynostosis, midface hypoplasia, and distinct facial anomalies. Affected infants may also have multiple joint contractures, bowing of the femora, long bone fractures, arachnodactyly, camptodactyly, renal agenesis, ambiguous genitalia, and cardiac defects. Morbidity and mortality are often related to choanal stenosis or atresia and respiratory distress [Antley and Bixler, 1975; Bottero et al., 1997; Chun et al., 1998; Reardon et al., 2000]. Although ABS has primarily been diagnosed after birth, recurrences have been recognized prenatally [Savoldelli and Schinzel, 1982; Schinzel et al., 1983; LeHeup et al., 1995; Kelley et al., 2002]. The cause of ABS is not yet known, and heterogeneity is likely. Based on reports of affected sibs born to unaffected parents and affected children born to consanguineous couples, ABS is considered an autosomal recessive condition in some families [Schinzel et al., 1983; Yasui et al., 1983; Suzuki et al., 1987; Feigin et al., 1995; LeHeup et al., 1995; Kelley et al., 2002]. Other modes of inheritance have also been suggested, including autosomal dominant inheritance associated with fibroblast growth factor receptor 2 (FGFR2) mutations [Chun et al., 1998; Tsai et al., 2001] and digenic inheritance where FGFR2 mutations are thought to account for the skeletal findings and different mutations, which cause altered steroidogenesis, are thought to contribute to genital abnormalities [Reardon et al., 2000]. Although the cause in most cases of ABS is considered to be genetic, early pregnancy exposure to high-dose fluconazole, an antifungal medication that inhibits the cytochrome p450 enzyme, 14-a-demethylase, is thought to be the cause of ABSlike skeletal characteristics in four reported cases [Lee et al., 1992; Pursley et al., 1996; Aleck and Bartley, 1997]. Because 14-a-demethylase (CYP51) catalyzes the conversion of lanosterol into principal intermediates of the distal portion of the cholesterol biosynthesis pathway, an abnormality in sterol synthesis was proposed as a potential cause of ABS in some cases. Kelley et al. [2002] demonstrated a defect in cholesterol biosynthesis at the level of CYP51 in a patient with ABS. However, no deletions, rearrangements, or mutations were found in any of the 10 exons or intron/exon boundaries of the CYP51 gene in their patient. Recently, a consistent and distinct urinary steroid profile has been reported in patients with genital ambiguity and/or an ABS-like phenotype. The term apparent pregnene hydroxylation deficiency (APHD) was coined to describe this condition [Shackleton and Malunowicz, 2003; Shackleton et al., 2004]. The defining biochemical characteristics of APHD include: (1) elevated urinary excretion of metabolites of pregnenolone

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and progesterone (pregnenediol and pregnanediol, respectively); (2) elevated metabolite levels associated with 17-a-hydroxylase deficiency (tetrahydrocorticosterone (THA), tetrahydro-11-dehydrocorticosterone (THB), 5-a-THB and 11oxo-pregnanediol); and (3) elevated metabolites characteristic of 21-hydroxylase deficiency (17-a-hydroxypregnanolone, pregnanetriol, and pregnanetriolone). Although the levels of metabolites associated with 17-a-hydroxylase deficiency and 21-hydroxylase deficiency were not generally above the normal physiological range, the ratio of their secretion relative to the secretion of urinary cortisol metabolites is significantly elevated. We report here the prenatal diagnosis and postnatal followup of sibs with classic ABS. Maternal serum screening at midgestation demonstrated undetectable unconjugated estriol (uE3) in each of the affected pregnancies, implying abnormal sterol or steroid metabolism in these children. In addition, the second affected child was shown postnatally to have both an abnormality in cholesterol biosynthesis at the level of CYP51 and the distinct urinary steroid profile associated with APHD. We propose that the prenatal and postnatal biochemical abnormalities have a common cause, which may play an important role in understanding the pathogenesis of this complex disorder and identifying candidate genes.

Fig. 1. Patient 1 as a neonate. Note the brachycephaly with marked midface hypoplasia, mild proptosis, bowing of the femora, ambiguous genitalia, and contractures at the elbows, fingers and knees.

CLINICAL REPORTS Patient 1 Patient 1 was born to a 26-year-old Caucasian mother and 23-year-old Caucasian father. The couple, their previous child, and the mother’s child from a prior relationship were all in good health. During the pregnancy, a maternal serum triple screen showed undetectable maternal serum uE3 at 16.6-week gestation. A repeat specimen confirmed this result. Sonographic studies detected multiple anomalies, including a flattened nasal bridge, low set ears, abnormal hand posturing, ambiguous genitalia, and bowed humeri, femora and tibiae. The family history was unremarkable and consanguinity was denied. Patient 1 was born at term weighing 3.2 kg. At delivery, he had significant airway obstruction due to choanal atresia, midface hypoplasia, and Robin sequence, which required intubation and a subsequent tracheostomy. A gastrostomytube was also required due to dysphagia. Bicoronal synostosis, frontal bossing, marked brachycephaly, midface hypoplasia, proptosis, radiohumeral synostosis, joint contractures, arachnodactyly, bilateral talipes, and bilateral femoral bowing with fractures were noted on physical examination (Fig. 1). Based on these findings, a diagnosis of ABS was made. Due to the presence of ambiguous genitalia, including micropenis and perineoscrotal hypospadias, endocrine and

genetic analyses were initiated. Baseline cortisol was normal (9.2 mg/dl) and was followed by a normal cortisol response (19.5 mg/dl) 1 hr after receiving 57.5 mg of ACTH. Chromosome analysis demonstrated a normal male karyotype (46,XY). Baseline FSH and LH levels, measured at 11 days, were both normal at 3.4 mIU/ml and <2.4 mIU/ml, respectively. An hCG stimulation test was performed at age 2 weeks using a single dose of 3,000 U/m2 of hCG (total units ¼ 1,350). Values were measured prior to and 3 days after stimulation. Increased levels of progesterone and 17-a-hydroxyprogesterone were noted before and after hCG stimulation. Following hCG stimulation steroid levels dropped with the exception of testosterone, which rose from 4.8 to 64 ng/dl (Table I). Based on the endocrine studies, a diagnosis of hypogonadotropic hypogonadism was made and the patient received two injections of testosterone enanthate (25 mg/IM) before the age of 5 months. Prior to the injections, the phallus was 3 mm in width, and following the injections the phallus grew to 13 mm in width and 32 mm in length. Postnatal complications included obstructive hydrocephalus, due to stenosis at the foramen magnum, and developmental delays. The hydrocephalus required placement of a ventriculoperitoneal shunt at approximately 3 months of age. Operations were performed to correct craniofacial abnormalities. At almost 2 years of age, global developmental delay was

TABLE I. Serum Steroid Levels (ng/dl) in Patient 1 at 2 Weeks of Age and 3-Day Post Stimulation With 3,000 U/m2 of hCG Steroid type Progesterone 17-a-hydroxyprogesterone Pregnenolone 17-a-hydroxypregnenolone DHEA Androstenedione Testosterone a

Baseline

Post hCG stimulation

Normal rangea

1529b 1000b

508b 263b

7–52 7–77

79 48c 12 64

36–763 50–760 6–68 20–50 (7 days) 60–400 (3 weeks)

Not measured 516 86 27 4.8c

Normal ranges are for infants 2 weeks of age except where specified. Values above the normal range. c Values below the normal range. b

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still present, although the patient was learning sign language and becoming mobile. The patient died at age 2 years when his tracheostomy became displaced. Patient 2 Patient 2 is the younger sister of Patient 1 and was recently reported as Case 4 in a series of patients with ABS and/or ambiguous genitalia [Shackleton et al., 2004]. During the mother’s fifth pregnancy, a maternal serum triple screen was performed at 14.4 weeks in which maternal serum uE3 was again undetectable. Sonography at 16 weeks gestation documented bowed femora and flexed fingers (Fig. 2). Chromosome analysis on amniocytes showed a normal female karyotype, 46, XX. FGFR2 mutation analysis did not identify a mutation in exons IIIa or IIIc. A 4-D ultrasound examination was performed at 21-week gestation. Brachycephaly, a prominent forehead, midface hypoplasia, flexed elbows and fingers, a small chest, a single umbilical artery, bowed femora, and rocker-bottom feet were noted (Fig. 3). During the pregnancy, the mother experienced facial edema and acne, which she also reported during her pregnancy with Patient 1. She denied any history of acne, facial edema, or other signs of androgen excess during her three earlier pregnancies, which had resulted in two healthy children with no signs of ABS and one miscarriage. A female infant was delivered at 392=7 weeks with a birth weight of 2.84 kg. Respiratory complications following birth required a tracheotomy. The prenatal diagnosis of ABS was confirmed based on the following physical findings: bicoronal synostosis, brachycephaly, severe midface hypoplasia, choanal atresia, moderate proptosis, bilateral radiohumeral synostosis, camptodactyly of fingers 2–5, varus bowing of femora, anteriorly placed anus, and mild hypoplasia of the labia majora without signs of virilization (Fig. 4). Baseline serum cortisol level was within normal limits (16.8 mg/dl, 5 days after posterior fossa decompression) at age 8 months, and there was no significant increase (18.6 mg/dl) 30 min after receiving 1 mg/m2 of ACTH. Urinary steroid profiling was performed at age 20 months using methods described previously [Shackleton, 1993; Caulfield et al., 2002]. Findings were consistent with APHD, and details are reported as Case 4 by Shackleton et al. [2004]. The concentrations of cholesterol, lanosterol, and dihydrolanosterol were measured in cultured EBV transformed lymphoblasts as described previously by Kelley et al. [2002].

Fig. 2. bowing.

Ultrasound of Patient 2 at 16-week gestation showing femoral

Fig. 3. Ultrasound of Patient 2 at 21 weeks gestation. a: Closeup of the arm shows radiohumeral synostosis. b: Frontal 4-D image. Note the bulbous nasal tip, prominent forehead, and flexed fingers. c: Facial profile illustrates brachycephaly and flattened midface.

After growth for 3 days in cholesterol-depleted medium, lymphoblasts from the patient accumulated abnormal amounts of lanosterol (0.459
0.136 mg/mg protein, N ¼ 4; normal controls 0.011
0.006 mg/mg protein, N ¼ 8) and dihydrolanosterol (0.411
0.079 mg/mg protein, N ¼ 4; normal controls 0.027
0.012 mg/mg protein, N ¼ 8). The level of cholesterol (9.30
3.26 mg/mg protein, N ¼ 4) was not significantly different from that of simultaneous controls (9.53


Fig. 4. Patient 2 at 9 months. a: Frontal view illustrates the typical facial appearance of Antley–Bixler syndrome including; brachycephaly, box-like forehead, midface hypoplasia, distinctive nose, flat nasal bridge, and small mouth. b: Profile view demonstrates the severity of the brachycephaly and midface hypoplasia. Also note the low-set, abnormally modeled ear. c: Right arm, demonstrating elbow contractures due to radiohumeral synostosis.

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3.44 mg/mg protein, N ¼ 8). These findings are similar to those previously reported in a male patient with ABS and ambiguous genitalia [Kelley et al., 2002]. The most recent developmental assessment at the age of 23 months showed delayed motor and speech development. Recent skills included pulling to a stand and cruising. The child communicates her needs mainly through the use of sign language and gestures. DISCUSSION Several reports in recent years have invoked inborn errors of sterol or steroid metabolism in the pathogenesis of ABS. Evidence for this has included an association of ABS with maternal luteoma [Roth et al., 2000; Warmann et al., 2000], 21-hydroxylase deficiency [Reardon et al., 2000; Warmann et al., 2000], first trimester exposure to fluconazole [Pursley et al., 1996; Aleck and Bartley, 1997], and lanosterol 14-ademethylase deficiency [Kelley et al., 2002]. Further support is provided by the report of a distinctive urinary steroid profile in seven patients who have a clinical diagnosis of ABS [Shackleton et al., 2004]. Although we were unable to perform a urinary steroid profile on Patient 1, endocrine studies showed similarities and differences when compared to results from other individuals with APHD reported by Shackleton and Malunowicz [2003]. Patient 1 had high serum levels of progesterone and 17-ahydroxyprogesterone, which is consistent with increased levels noted in patients with APHD. However, unlike five patients with APHD in which serum levels were reported, 17-hydroxypregnenolone was not elevated in Patient 1. Low serum testosterone in Patient 1 is consistent with previous reports of APHD. However, unlike patients reported with APHD where testosterone failed to respond to hCG, the testosterone level of Patient 1 increased normally in response to hCG stimulation. Individuals with APHD typically show low-normal cortisol levels that respond poorly to ACTH stimulation. However, cortisol levels in Patient 1 were normal and showed a normal response to ACTH stimulation. The reason for the differences in serum steroid levels between Patient 1 and other patients with APHD is uncertain. However, it probably represents variable expression of this trait. Further studies including larger numbers of patients will be needed to define the degree of variability seen in APHD. There are some well-known examples of genetic conditions where abnormalities in steroidogenesis lead to ambiguous genitalia. Deficiencies in the enzyme CYP17 results in ambiguous genitalia in males, while the most common form of congenital adrenal hyperplasia (CAH), caused by deficiency in CYP21, is associated with masculinization of the external genitalia in females. A deficiency in 3-b-hydroxysteroid dehydrogenase is responsible for a rare form of CAH that leads to ambiguous genitalia in males, due to decreased testosterone levels, and virilization in females, due to increased levels of weak androgens. Unlike these well-known conditions, the primary underlying defect responsible for ambiguous genitalia in ABS remains uncertain. Although low testosterone may contribute to ambiguous genitalia in Patient 1 and other males with APHD, it is unlikely to be a complete explanation. Additional complications arise when considering why a number of females with ABS have ambiguous genitalia, while others, including Patient 2, do not. Further characterization of the underlying steroid defect(s) in patients with ABS may not only answer these questions but improve our understanding of the mechanisms that cause ambiguous genitalia. The observation that maternal serum uE3 was undetectable at mid-trimester in both of the cases reported here provides additional evidence that errors in steroid metabolism contribute to the ABS phenotype in some cases. Although low

levels of maternal serum uE3 have primarily been used to calculate fetal risks for trisomy 18 and Down syndrome [Canick et al., 1988, 1990; Wald et al., 1988; Crossley et al., 1993], extremely low or undetectable levels of uE3 have been associated with a variety of errors in steroidogenesis or cholesterol synthesis. These include steroid sulfatase deficiency [Taylor and Shackleton, 1979; Bartels et al., 1994; Zalel et al., 1996; Glass et al., 1998], congenital adrenal hypoplasia [Peter et al., 1996], Smith–Lemli–Opitz syndrome (RSH/ SLOS) [Bradley et al., 1999; Kratz and Kelley, 1999; Shackleton et al., 2001], and aromatase deficiency [Shozu et al., 1991]. The cases reported here suggest that the diagnosis of ABS should also be considered in pregnancies with low or undetectable maternal serum uE3. Interestingly, the mid-trimester maternal serum uE3 level for RSH/SLOS pregnancies has been shown to correlate directly with the severity of the cholesterol synthesis defect and typically is lower than 0.2 MoM only in patients found to have the lowest cholesterol levels at birth [Kratz and Kelley, 1999]. In our cases, it is more likely that undetectable maternal serum uE3 levels are caused by an apparent block in steroid biosynthesis, as in steroid sulfatase deficiency, rather than diminished cholesterol synthesis, as in RSH/SLOS. This is supported by the finding that cholesterol levels in lymphoblast cells from Patient 2 were not significantly different from those of controls, whereas cholesterol levels in RSH/SLOS lymphoblasts fall below levels in normal controls by the third day, even for most of the mild RSH/SLOS patients [Kelley, unpublished observations]. Additional support comes from findings of normal blood cholesterol levels in other ABS patients [Kelley, unpublished observations]. Further insight into the pathogenesis of ABS comes from the combination of undetectable maternal serum uE3, the distinct urinary steroid profile, and an abnormality in sterol synthesis at the level of 14-a-demethylase in a single patient with a similarly affected sibling. The most parsimonious explanation is that a single autosomal recessive defect is causing disturbances in both steroid and sterol pathways in our patients. Shackleton and Malunowicz [2003] suggested that genes encoding essential redox partners or allosteric effectors involved in steroid hydroxylation were potential candidate genes responsible for abnormalities seen in APHD. NADPH cytochrome P450 reductase (CPR) and cytochrome b5 (CYb5) appear to be likely candidates in our patients, primarily because of their roles in both steroid and cholesterol biosynthesis. CPR is a membrane-bound flavoprotein that receives electrons from NADPH and contributes them to a number of cytochrome P450 enzymes. CPR is required for the 17-ahydroxylase activity of the cytochrome p450 enzyme, CYP17, which is responsible for the conversion of pregnenolone to 17-ahydroxypregnenolone and progesterone to 17-a-hydroxyprogesterone. A partial reduction in 17-a-hydroxylase activity, resulting from a mutation in CPR, may explain the elevated serum progesterone level measured in Patient 1 and the increased urinary progesterone and pregnenolone metabolites observed in Patient 2 (Fig. 5). In addition, reduction in 17-ahydroxylase activity could also explain the relatively increased levels of metabolites associated with 17-hydroxylase deficiency. CPR contributes electrons to CYP21, which is responsible for the multi-step production of cortisol from 17-ahydroxyprogesterone. Thus, a mutation in CPR could be responsible for the elevated serum level of 17-a-hydroxyprogesterone in Patient 1 and the higher relative amount of urinary metabolites associated with 21-hydroxylase deficiency in Patient 2 (Fig. 5). CYb5 is a protein that has long been thought to aid in a number of cytochrome p450 reactions. Specifically it has been shown to facilitate the 17,20-lyase activity of CYP17 by allosterically promoting the interaction of CPR and CYP17 [Auchus et al., 1998]. Furthermore, CYb5 appears to influence

Steroid & Sterol Abnormalities in ABS

Fig. 5. Principal intermediates of steroidogenesis illustrating the location of multiple partial biochemical blocks that would presumably cause increases in pregnenolone, progesterone, and 17-a-OH-progesterone (bold) along with relative increases in urinary metabolites associated with 17- and 21-hydroxylase deficiency compared to cortisol metabolites (not shown). Cytochrome P450 reductase (CPR) plays a role at each of the enzymatic steps catalyzed by CYP17 and CYP21. Cytochrome b5 (CYb5) facilitates the 17,20lyase activity of CYP17 and could potentially influence other steps. Note: Partial blocks are presumed based on results demonstrating the unique urinary steroid profile of APHD in Patient 2 as well as serum steroid levels in Patient 1.

the regional control of 17-a-hydroxylase [Mapes et al., 1999; Miller and Auchus, 2000]. In addition, there is some evidence that CYb5 may play a role in both 17-hydroxylase and 21hydroxylase activities in guinea pigs [Kominami et al., 1992]. A defect in either CPR or CYb5 could also explain the undetectable maternal serum uE3 in our patients. Formation of maternal serum uE3 is described below and illustrated in Figure 6. During pregnancy, the placenta makes pregnenolone and upon transfer to the fetus, a sulfate group is added. The fetal adrenal gland then converts pregnenolone sulfate to dehydroepiandrosterone sulfate (DHEAS) using the 17-hydroxylase/17,20-lyase activities of CYP17. Recently, Rehman et al. [2003] demonstrated that cytochrome b5 and CPR were expressed 2.3- and 2.0-fold higher in the human fetal adrenal glands compared to adult adrenal glands. Based on their observations, the authors concluded that CYb5 and CPR may

Fig. 6. The main intermediates in the production of maternal serum uE3 during pregnancy. Undetectable levels of maternal serum uE3 during pregnancy provides evidence that this pathway was interrupted in the sibs reported here. CPR and CYb5 appear to play an important role in the reactions catalyzed by CYP17 in the human fetus [Rehman et al., 2003]. The expression of human fetal CYP3A7 and human CPR was sufficient for the CYP3A7-dependent 16-a-hydroxylation of DHEA and DHEAS when coexpressed in insect cells, and CYb5 may be necessary for maximal activity of CYP3A7 [Ohmori et al., 1998].

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upregulate 17-a-hydroxylase/17,20-lyase activities thereby increasing DHEAS production in the human fetal adrenal gland. After DHEAS is produced, it is converted to 16-ahydroxy-DHEAS via the 16-hydroxylation activity of CYP3A7 in the fetal liver. Ohmori et al. [1998] have shown that coexpression of human fetal CYP3A7 and CPR in insect cells was sufficient for 16-hydroxylation of DHEAS, but the addition of CYb5 was necessary to obtain maximal activity of CYP3A7. The accumulation of lanosterol and dihydrolanosterol in lymphoblasts grown in the absence of cholesterol can also be explained by defects in either CPR or cytochrome b5 (Fig. 7). Specifically, CYP51 and CPR compose the lanosterol 14-ademethylase protein complex and cytochrome b5 has been shown to significantly increase CYP51 activity in humans [Lamb et al., 2001]. Abnormalities of bone formation and limb patterning are common findings among disorders of cholesterol synthesis [Kelley, 2000]. None of the mechanisms by which these disorders are thought to cause skeletal abnormalities can easily explain the distinctive skeletal anomalies of ABS [Kelley and Herman, 2001; Cooper et al., 2003]. However, the finding that inhibition of squalene synthase, the enzyme just preceding CYP51 in cholesterol synthesis, leads to an accumulation of compounds involved in the isoprenylation of signaling proteins, including those regulating osseous remodeling, may help explain the skeletal defects and other malformations characteristic of ABS [Vaidya et al., 1998]. Defects in CPR may also play a role in the skeletal anomalies present in individuals with ABS and no identifiable FGFR2 mutation. Rudimentary evidence supporting a role of CPR in skeletal development comes from a study of CPR expression in mouse limb formation demonstrating elevated levels of CPR mRNA transcripts in mesenchymal cells where two or more precartilages come together to form joints. Levels of CPR mRNA were particularly prominent in mesenchymal cells between the humerus, ulna and radius, and between the future phalanges and metacarpal bones [Keeney and Waterman, 1999]. Based on these findings, the authors conclude that CPR may play an unidentified function in the early development of joints and in the formation of precartilage. While it is clear that both CPR and CYb5 are important to the steroid and sterol metabolic pathways, we believe mutations in CPR are more likely to lead to ABS. There are two reasons why CYb5 is perhaps a less likely candidate: (1) When expressed in yeast with human CYP17 and CPR, CYb5 does not appear to significantly alter 17-a-hydroxylase activity [Geller et al., 1999] and (2) the only patient reported with a mutation

Fig. 7. Evidence for a partial biochemical block in sterol synthesis at the level of 14-a-demethylase comes from the finding of significantly increased levels of lanosterol and dihydrolanosterol (bold) when lymphoblasts from Patient 2 were grown in the absence of cholesterol; 14-ademethylase activity requires both CYP51 and CPR, and its activity is increased significantly by CYb5.

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in CYb5 was a male pseudohermaphrodite with congenital methemoglobinemia [Hegesh et al., 1986; Giordano et al., 1994]. Although this patient was initially reported to be female due to ambiguous genitalia, no other findings consistent with a diagnosis of ABS or APHD were reported. To our knowledge, no patient has been reported with a mutation in CPR. In summary, the findings reported here strongly suggest that there is a disturbance in both steroidogenesis and sterol metabolism in at least some patients with ABS. Documentation of undetectable mid-trimester maternal serum uE3, the distinct urinary steroid profile diagnostic of APHD, and an abnormality in sterol synthesis at the level of 14-a-demethylase in a single patient with ABS is consistent with the hypothesis that the underlying defect occurs in an element, such as NADPH CPR or cytochrome b5, that is common to each of these pathways.

Giordano SJ, Kaftory A, Steggles AW. 1994. A splicing mutation in the cytochrome b5 gene from a patient with congenital methemoglobinemia and pseudohermaphrodism. Hum Genet 93:568–570.

ACKNOWLEDGMENTS

Kelley RI, Kratz LE, Glaser RL, Netzloff ML, Wolf LM, Jabs EW. 2002. Abnormal sterol metabolism in a patient with Antley-Bixler syndrome and ambiguous genitalia. Am J Med Genet 110:95–102.

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