UN.

CHEM.

40/11,

2099-2103

(1994)

ongenital Adrenal Hypoplasia, Duchenne Muscular )eficiency: Importance of Laboratory Investigations 3ene Deletion Syndrome
avid onia E. C. Cole,3 Salisbury
Lorne

Dystrophy, and Glycerol Kinase in Delineating a Contiguous

A.

A.

Clarke,

D.

Christie

Riddell,

Karen

Samson,

William

K. Seltzer,2

and

Ve describe an infant with adrenai insufficiency who was ubsequently diagnosed with Duchenne muscular dystrophy DMD) and hyperglycerolemia due to glycerol kinase deliiency. Karyotyping showed a deletion on the short arm of the chromosome (p21.1 to p22.1). Molecular mapping revealed iat the deletion extended from the 3 end of the DMD gene a site telomeric to the loci for X-linked congenital adrenal ypoplasia and glycerol kinase deficiency. These results are iagnostic for an Xp21 contiguous gene deletion synrome-so named because the deletion manifests as a disnctive cluster of otherwise unrelated single-gene disorders in ie same indMdual. The Xp21 syndrome should be considred inany infant with adrenal insufficiency. Measurement of erum tnglycendes (without glycerol blanking) and creatine iriase activity are simple screening tests that may facilitate any diagnosis and appropriate genetic counseling about sks of recurrence in subsequent offspring. idexlng Terms: adrenal insufficiency/chromosomal
able
disorders/pediatric chemistry

nase (GK EC 2.7.1.30) deficiency molecular studies used to confirm the deletion in this child. Case
cesarean

(7). We also describe and map the extent

the of

Report

mapping/her-

infant was delivered at 39 weeks gestation by section without significant perinatal complications. He first came to medical attention at age 16 days because of feeding difficulties, dehydration, and a weight loss of 680 g from a birth weight of 2750 g. His response to rehydration was hyponatremia (128 mmol/L) and hyperkalemia (9.6 mmol/L), prompting a working diagnosis of CAH. Subsequent investigation revealed decreased plasma aldosterone [110 pmol/L (reference range 970-3610 pmol/L)], decreased plasma dehydroepiandrostenedione sulfate [0.2 tmol/L (4.3-9.5 moVL)], and normal plasma 17a-hydroxyprogesterone [11.4 nmol/L (<30 nmol/L)], thereby making the 21hydroxylase-deficient form of CAll unlikely. An ACTH
test (with 0.125 mg of intravenous cosynprovoked an increase in serum cortisol from 419 nmoIfL in 60 min (reference range: peak serum >550 nmolfL). Serum testosterone was 5.9 nmoLL (reference range for children, >0.2 nmol/L). Serum fofficle-stimulating hormone (FSH) was 5.4 lU/L (reference range for infants, 1-12 lU/L), and serum luteinizing hormone (LH) concentrations at ages 16 and 38 days were 9.4 and 30.6 lU/L, respectively (reference values for infants, 22 13 lU/L). Fludrocortisone treatment corrected the electrolyte abnormalities, and the patient improved and was discharged home. He was seen periodically for management of his adrenal insufficiency. Over the next few months, the ACTH-stimulated concentrations of plasma cortisols declined. At age 9 months, his baseline and ACTH-stimulated cortisol concentrations were 181 and 173 nmol/L, respectively, confirming the diagnosis of CAH. Subsequently, cortisone acetate was added to the treatment regimen, leading to further improvement in motor strength and well-being. Mineralocorticoid and glucocorticoid replacement has been continued since. At 13 months, the child was noted to have an exotropia and mild hypertelorism, but his vision was not assessed in detail. Skin pigmentation was normal but bilateral cryptorchidism was observed. At age 2 years, surgical exploration revealed intraabdominal, atrophic stimulation

A male

Coincident expression of otherwise unrelated inborn erof metabolism may oocur as a result of a large, mu!iple-gene deletion, referred to as a contiguous gene deleion syndrome (1-4). The study of affected patients is tseful in localizing and determining the order of specific nes. The routine clinical laboratory may play an imporant part in identifying these associations and signifiantly influence the quality of care (5). We describe an nfant with congenital adrenal hypoplasia (CAH)4 in rhom suspicion of Duchenne muscular dystrophy (DMD) ad to the diagnosis of an Xp21 contiguous gene deletion yndrome (6) and identification of coincident glycerol kiors

tropin) to 737 cortisol

of Pediatrics and Pathology, Dalhousie UniverNova Scotia, Canada B3H 3G9. Department of Pediatrics, University of Colorado School of ledicine, Denver, CO 80262. 3Mdress for correspondence: Department of Clinical Biochemistry, rniversity of Toronto, 100 College St., Rm. 415, Toronto, Ontario, Canda M5G 1L5. Fax 416-978-5650, E-mail cole@madac.med.utoronth.ca Nonstandard abbreviations: DMD, Duchenne muscular dystrohy; GK, glycerol kinase; PCR, polymerase chain reaction; ACTH, drenocorticotropic hormone (corticotropin); GnRH, gonadotropineleasing hormone (gonadoliberin); FSH, follicle-stimulating ormone (follitropin); LH, luteinizing hormone (lutropin); CK, cretine kinase; CAll, congenital adrenal hyperplasia; AHC, -linked congenital adrenal hypoplasia; and FISH, fluorescent in itu hybridization. Received March 4, 1994; accepted August 12, 1994
Departments itr, Halifax,

CLINICAL

CHEMISTRY,

Vol. 40, No. 11,

1994

2099

testes 0.3 and 0.4 cm in diameter. After the removal of all testicular tissue, baseline and gonadotropin-releasing hormone (GnRH)-stimulated concentrations of FSH and LH were undetectable, suggestive of hypogonadotropic hypogonadism (8). Although initially this child was thought to be hypertonic and visually impaired, his vision improved while

his muscle tone decreased. Developmental delay and growth failure were mild but persistent and progressive. At age 2.7 years, his call muscles were noted to be enlarged. Serum total creatine kinase (OK) activity was 21 150 U/Land a skeletal muscle biopsy was histopathologically consistent with DMD. The possibility was considered that he might have an unusual X-linked disorder of retinal rod cells, called incomplete stationary night blindness (akin to Aland Island disease or Forsius-Eriksson-Miyake syndrome) (9, 10). However, evidence now suggests that deletions in the dystrophin gene can result in failure to express a retina-specific leading to characteristic clinical and electroretinographic changes in vision (11). At this point, the diagnosis of a contiguous gene deletion syndrome was entertained, and additional studies were undertaken to confirm the diagnosis.
dystrophin protein,

adopted out, was reported as normal, but further fo] low-up was not available. Glycerol kinase deficiency. Plasma triglycerides, mea sured by dry film methodology with dye-coupled enzy matic detection of glycerol without blanking (13), wer 4.3 1.1 mmol/L (mean SD, n = 3; reference rang 0.24-1.7 mmol/L). Serum cholesterol was 4.6 0. mmol/L (reference range, 2.8-5.2 mmol/L), and result of lipoprotein electrophoresis were unremarkable. Un nary glycerol concentration, measured by gas chroma tography-mass Mamer, Montreal, spectrometry QU), was (5) (courtesy of 4910 mg/g creatinine 0. A
(rel

erence range, <2 mg/g creatinine). GK activity wa measured radiochemically in lymphocyte extracts a previously described (14, 15), by using [U-4C}glycero as substrate and quantifying the formation product. Assay reactions (100 L) contained sample sonicate (50 g of protein), 100 mmoIJL (pH 7.4 at 37#{176}C), 1.0 mmol/L EDTA, 4 mmol/L 20 mmol/L 2-mercaptoethanol, mmol/L ATP, and 50 mol/L 1) indicate that the activity the controls cient patients of labelei 20 iL o Tnis-HC
MgSO4

form

of the

DM1)

6.25 mmol/L NaF, glycerol. The results (Tabb in our patient was <10% a

Methods

and Results
Chromosomes from peripheral blood examined by routine metaphase banding as normal (46,XY). Repeat karyotyping chromosomes (-550-band interstitial deletion of the X

Karyotyping. lymphocytes were reported

and matched that seen in other GK-defi assayed by the same technique. Molecular analysis. Genomic DNA was extractei from peripheral lymphocytes by the salting-out metho of Mifier et al. (16). The DNA was analyzed either b multiplex polymerase chain reaction (PCR) (17, 18) 0: by standard Southern transfer procedures (19) witl both flanking and intragenic probes. As shown in Fig. 2 the most centromeric intragenic probe that hybridize to the patients DNA was the cDNA probe, 44-1, locali.ze( to the midregion of the DMD gene. Neither the prob detecting the 3 end of the DM1) gene (J66/DXS268) no; that for the closely flanking region (07/DXS 28) gave i hybridization signal. The closest telomeric probe tha gave a positive (albeit weak) signal was the 99-6/DXS4: probe. This is a consistent feature of the Xp21 deletioi patients reported to date (20). Genomic DNA was amplified in PCR-multiplex reac tons, subjected to agarose gel electrophoresis, and visual ized by staining with ethidium bromide. The gels (Fig. 3 reveal the presence of appropriately sized amplicons up ti and including DMD exon 52, but no product was detecte for the next axnplicon within exon 60. This is consisten with the Southern transfer evidence for a 3 dystrophii deletion, and localizes the centromeric start of the deletioi to a site between exon 52 and the marker, J66/1)XS268 which maps to a region between exons 54 and 55.
syndrome

and examination of extended resolution) revealed a small

chromosome in the p21.3 region (Fig. 1). Because the child had been adopted, the mother could not be tested. The prophase karyotype of a biological sister, also

AHC GKD DMD XK CYBB

OTC
11.4

u-a u-a

-3
II II 11.1. II l

3
211

l.a

q
27

CONTROL

S.S.

Table
from the

1. Leukocyte

glycerol

kinase

actIvIty.
of protein Range 33-34

GK, mU/g

Fig. 1. Prometaphase G banding of the X chromosomes patient (S.S.) and a control.

An idiogram of the X chromosome shows the centromere (hatched line) and region (Xp21.1 to Xp22.1) that may be deleted in Xp21 contiguous deletion syndromes. The arrows indicate the genes shown by molecular means to be deleted in our patient Standard abbreviations (McKusick catalog (12)1, beginning from the cenlromere, are: OTC, omithine carbamyltransferase; cVBB, chronic granulomatous disease; XK McLeod phenotype; DMD, dystrophin gene; GKD, glycerol kinase deficiency; and AHC, congenital adrenal hypoplasia

Patienta Concurrent negative controla Accumulated negative controls (n

MeanSD 34

392
= 10) 455 35

31 4-46(
83 232-45

Accumulated
a

positive controls
determinations.

(n = 4)

19

20-63

For triplicate

2100

CLINICAL

CHEMISTRY,

Vol. 40, No. 11,

1994

CSS

CSS

SS

C SS

Size(Kb) -21.7

some may be so small as to be undetectable by either of these methods; (c) specific aspects of these syndromes are clearly recognizable as single-gene mendelian traits; and (d) the spectrum of abnormalities in affected patients can be deduced from the mutant phenotypes associated with the single-gene mutations involved. Contiguous gene syndrome loci identified to date are not found randomly scattered throughout the human gename but are localized cytogenetically to regions associated with a consistent phenotype that is clinically recognized. A contiguous gene syndrome, if localized to a region on an autosome (nonsex chromosome), is not commonly accompanied by a characteristic metabolic profile. This follows from the rule that few enzymatic deficiencies are expressed as dominant conditions. Autosomal synof ge-

-1.6 -1.4

-0.9 :ig. 2. Composite (44-1) (J66) of autoradiograms

(Cl) (99-6)

for Southern

transfers

omic DNA hybridized to labeled probes (in parentheses) spanning e Xp21 region. hown are size ladder markers (M), and samples from a control (C) paired iith the patient (SS) and the four probes, ordered (left to right) in the 5 entromenc) to 3 (telomenc) direction. The restriction fragment pattem for the 4-1 probe is unaltered, but the patients DNA does not hybridize to either of ie probes J66 or C7. Two fragments of -1.0 and 20 kb hybridize faintly but istinctly to the 99-6 probe.

MultIplex

Multiplex

II

:ig. 3. Agarose gel electrophoresis

i

of multiplex PCR

products.

each reaction, the patients profile (SS) is compared with female (C9) and iale (Ce) control DNA amplification products and a 100-bp ladder of marker INA (M) and a blank (Bik). The exonic location of the amplified products is sticated, and the absence of exon 60 product in our patient is marked with a.

)lscussion
The term contiguous gene syndrome was first used iy Schmickel (1) in 1986 to describe patients with muliple single-gene disorders originating from deletion or Luplication of a chromosomal segment but not detected iy routine karyotyping. From a genetic standpoint, the ontiguous gene syndromes constitute a class of mutaions falling somewhere between the large chromosomal Leletions and the mendelian disorders that result from a ingle-gene deletion (3, 4). The contiguous gene syn[romes are characterized by the following (4): (a) they ire usually sporadic (but can be familial) and are inherted in a dominant fashion; (b) the cytogenetic abnorrialities may be detectable by high-resolution karyotyprig or fluorescent in situ hybridization (FISH), although

dromes are therefore identified by their clinical presentation and usually require the expert opinion of a dysmorphologist for definitive diagnosis. Examples include the Langer-Gideon syndrome, the Miller-Dieker lissencephaly syndrome, the Smith-Magenis syndrome, and the Xp21 syndrome of choroideremia, deafness, clefting, and mental retardation (3, 4). For several reasons, the diagnosis and delineation of contiguous gene deletion syndrome associated with Xp21 is more dependent on laboratory studies. First, the Xp21 deletion syndrome, like the other contiguous gene deletion syndromes, occurs sporadically, so a significant family history is usually not obtained. Second, the extent of the deletion itself is unique to the patient, so the resulting phenotype is more heterogeneous than the constituent single-gene deletion disorders. Third, the dysmorphic features are minimal and nonspecific (21). Fourth and finally, the associated inborn errors of metabolism are not always easily recognized by their clinical presentation. Although there is stifi some doubt as to the number of genes that may be encompassed by the Xp21 syndrome, it is clear that the deletion most frequently affects the X-linked congenital adrenal hyperplasia (AHC), GK, and dystrophin (DMD) loci (Fig. 1). Those patients with deletions that extend proximally or 5 to the DM1) locus, including the determinant of the erythrocyte MacLeod phenotype, chronic granulomatous disease, and the ornithine transcarbamylase enzyme of the urea cycle, have tended to be female carriers. In males with the Xp21 syndrome, AHC is usually the first condition to be manifest. For the majority of patients who present with hyponatremia and hyperkalemia, the more common condition of CAH can be excluded by measuring 17a-hydroxyprogesterone and conducting an ACTH stimulation test and an assay of plasma aldosterone. Rapid diagnosis is important, since steroid replacement may be lifesaving, but suspicion of AHC should be sufficient indication for further laboratory investigations. The differential diagnosis includes X-linked adrenoleukodystrophy and an ACTH-resistance syndrome caused by an ACTH receptor defect (22). The first can be ruled out by screening for increased plasma concentrations of very-long-chain fatty acids, which are a characteristic feature of X-linked CLINICAL CHEMISTRY, Vol. 40, No. 11,
1994

2101

and other inborn errors of peroxdegradation of these fatty acids. The other condition, resistance to ACTH, is not associated with mineralocorticoid deficiency and therefore may be excluded on clinical grounds, but confirmation may require determination of plasma aldosterone. As an isolated condition, AHC is a rare disorder with a degree of adrenal dysfunction that varies dramatically between individuals and with age (22-25). Its pathogenesis is still obscure. For some time, it has been recognized as usually associated with a form of hypogonadotropic hypogonadism. In male infants, an early manifestation may be small or cryptorchid testes; this is another feature that distinguishes AHC from CAN, the latter being associated with normal or hypertrophic genitalia. Some authors have speculated that another gene locus mapping distal to AHC may exist, associated solely with the hypogonadotrophic hypogonadism phenotype (24). But it is also possible that AHC and hypothalamic hypogonadism are alternative expressions of different mutations within the same locus, akin to the situation for DMD and Becker muscular dystrophy and X-linked dilated cardiomyopathy (26). Overall, the most likely
isome-mediated

adrenoleukodystrophy

constituen before ag 3 years. Thus, laboratory medicine plays a crucial rol in identification of this condition as part of an Xp2 syndrome. In patients with suspected AHC, screenin, a serum CK determination. Di be identified by the artifactuall increased concentrations of serum triglycerides associ ated with hyperglycerolemia. Parenthetically, we not that GK deficiency is another disorder that should b kept in mind as a cause of an inexplicable increase c triglycerides in assays unblanked for glycerol (27-29) Although large chromosomal deletions may be de tectable by routine karyotyping, a positive biochemica screen in this type of patient should be an indication fo higher-resolution banding in search of smaller dele tions. McCabe et al. (20) identified Xp21 deletions by bivariate-flow karyotyping method, using a double staining technique coupled to a dye-sensitive eel sorter, but not all microdeletions would necessarily b detectable by this method. In a more general way for DMD requires ficient GK activity only may

DMD is decidedly single-gene disorders,

the

most common of the but it rarely manifests

explanation pogonadism

is that the phenotypes

AHC and hypogonadotropic are both related to a single

hyun-

Table 2. Selected
Gene

contiguous
Congenital

gene deletion
Associated

syndromes.

deletion

[locatIon]

disorders

derlying primary disorder that progressively disrupts hypothalamopituitary and adrenal function. As evidence for this, Kletter et al. (22) point to the similar cytomegalic changes found on histopathologic exainination of both pituitary and adrenal glands. They also note the parallel decline in function of both glands in most reported cases, including three of their own. Findings in our patient substantiate this progressive decline in function. Normal values for serum gonadotropins, testosterone, and peak cortisol indicate significant residual glandular function in the first month postpartum. By age 2 years, however, there was no detectable adrenal response to ACTH, and the gonadotropin stimulation test was flat. After bilateral orchidectomy, gonadotropins remained undetectable, despite the stimulatory feedback effect that the agonadal state would normally hypothalamopituitaiy axis. Because other patients have had similarly variable responses to various GnRH-stimulation tests, the question of whether the hypogonadotropic state originates with the pituitary gonadotroph cells or also involves the hypothalamus and its release of GnRH is still unresolved (22, 23, 25). Further clinical studies are likely to be instructive, but clinical and molecular genetic analyses of new cases may be the fastest route to settling the controversy. The variable natural history of GK deficiency as an isolated condition poses similar problems. GK deficiency has been identified in some children with vomiting, hypoglycemia, and somnolence, but is also seen in adults without any clinical symptoms (6). In these isolated GK-deflcient patients, the possible contribution of an undetected contiguous gene deletion cannot be ruled out. Indeed, from the available data, the developmental delay found in most Xp21 syndrome patients cannot yet be attributed to any one specific locus.
exert on the

[Xp21 J

adrenal hypoplasiaa

Glycerol kinase deficiency with

hyperglyerolemiaa Duchenne muscular dystrophya
McLeod Omithine Chronic phenotypea
transcarbamylase

deflciencya diseasea

granulomatous

[Xp22.3]

?Hypogonadotrophic hypogonadism ?intermittent stationary night blindness X-linked ichthyosis with steroid sulfatast
deflciencya

Kaliman syndrome
Chrondrodyspiasia punctata Mental retardation and short stature Ocular albinism Hypoplastic parathyroids with
hypocalcemia8

DiGeorge sequence [22q1 1]C

Thymic hypoplasia
Iymphopeniaa Facial dysmorphism

with T-cell

Alagille syndrome or
arteriohepatic

Congenital heart disease Biliary duct hypoplasia with chronic cholestasisa

dysplasia

120P11

Facial dysmorphism
Vertebral anomalies Peripheral pulmonic stenosis or
hypoplasia

Li 6p1

33]10

aThalassemiaa
gene deletion

8Contiguous

Mental retardation syndromes with associated

findings.

features

that pre

sent with characteristic laboratory b Seen only in a female carrier.

The diGeorge sequence is associated with a wide variety of conditiooe hereditary and acquired (2). Whether all cases are the consequence of microdelection has not been determined. d Summary and review of recent literature can be found in McKusick (12) also available on-line. #{149} is also an X-linked form of this disorder, which has not been we There characterized.

2102

CLINICAL

CHEMISTRY,

Vol. 40, No. 11,

1994

FISH has become the method of choice for identifring tered hybridization of molecular probes to specific ites in metaphase chromosome spreads, and has been portant in mapping various contiguous gene deleions

(30);

however,

both

procedures

are

technically

emanding.

In our patient, the Xp21 deletion was demonstrable th standard high-resolution karyotyping, but direct olecular mapping did not detect altered size fragments hat might have pinpointed one of the breaks. Thus, our suits provide only an upper limit on the physical size of he deletion. Techniques for assessing the extent of the eletion will improve as the number of markers in the 2 1-23 region increases, but the exact determination of e breakpoint would require further mapping and largee sequencing from the nearest flanking markers. Although this childs adrenal insufficiency can be ;reated by hormone replacement, the long-term progno;is is otherwise guarded. However, confirmation and lelineation of the extent of the Xp21 deletion by labo-atory studies allows for early screening of families to Ldentify possible carrier status and permit preventive ounseling and prenatal diagnosis with respect to the risks of recurrence in subsequent male offspring (31). The Xp21 syndrome is one of at least five different ontiguous gene deletion syndromes that can be identibed or suspected by laboratory testing (Table 2). The Kp22.3 syndrome, which maps to a more distal region on he short arm of the X chromosome, may be dig;inguished biochemically by the presence of steroid sulatase deficiency as one of a cluster of single-gene disorlers. The other conditions may have laboratory findings that are nonspecific but characteristic enough to raise the Lndex of suspicion when other clinical features are pretent. Contiguous gene syndrome delineation is still very nuch in its infancy, and there will undoubtedly be other onditions for which laboratory tests contribute sigriifiantly to the diagnostic process (2). eferences
RD. Contiguous gene syndromes: a component of ecognizable syndromes. J Pediatr 1986;109:231-41. i. Cohen MM Jr, Cole DEC. Origins of recognizable syndromes: ttiologic and pathogenetic mechanisms and the process of synIrome delineation. J Pediatr 1989;115:161-4. I. Ballabio A. Contiguous deletion syndromes. Curr Biol 1991;1: 1. Schmickel 15-9.

disease to Xp21 between DXS67 (B24) and Duchenne muscular dystrophy. Am J Hum Genet 1990;47:795-801. 10. Pillers DM, Seltzer WK, Powell BR, Lewis RA, Ray PN, Bulman DE, et al. Ophthalmologic findings in complex glycerol kinase deficiency: electroretinogram suggests congenital stationary night blindnesWAland Island eye disease [Abstract]. Pediatr Res 1991;29:133A. 11. Pillers DM, Sigesmund DA, Ray PN, Musarella MA, Tramblay F, Seltzer WK, et al. Genotype-phenotype correlations identified by electrophysiology of the retina in Duchenne and Becker muscular dystrophy patients [Abstract]. Am J Hum Genet 1993;

53:146. 12. McKusick VA. Mendelian inheritance in man, 10th ed. Baltimore: Johns Hopkins Univ. Press, 1992. 13. Nfigele U, Hfigele EO, Sauer G, Wiedemann E, Lehmann P, Wahlefeld AW, Gruber W. Reagent for the enzymatic determination of serum total triglycerides with improved lipolytic efficiency. J Clin Chem Clin Biochem 1984;22:165-74. 14. Seltzer WK, McCabe ERB. Human and rat adrenal glycerol kinase: subcellular distribution and bisubstrate kinetics. Mol Cell
ERB. Subcellular distribution and hiand particulate-associated bovine wirenal glycerol kinase. Mol Cell Biochem 1984;64:51-61. 16. Miller SA, Dykes DD, Polesky HF. A salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. 17. Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988;16: 11141-56. 18. Beggs All, Koenig M, Boyce FM, Kunkel LM. Detection of 98% of DMDIBMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45-8. 19. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mo! Biol 1975;98: 503-17. 20. McCabe ERB, Towbin JA, van den Engh G, Trask BJ. Xp21 contiguous gene syndromes: deletion quantitation with bivariate flow karyotyping allows mapping of patient breakpoints. Am J Hum Genet 1992;31:1277-85. 21. Wise JE, Matalon R, Morgan AM, McCabe ERB. Phenotypic features of patients with congenital adrenal hypoplasia and glycerol kinase deficiency. Am J Dis Child 1987;141:744-7. 22. Kletter GB, Gorski JL, Kelch RP. Congenital adrenal hypoplasia and isolated gonadotropin deficiency. Trends Endocrino! Metab 1991;2:123-8. 23. Kruse K, Sippeli WG, Schnakenburg KV. Hypogonadism in congenital adrenal hypoplasia: evidence for hypothalamic origin. J Clin Endocrinol Metabol 1984;58:12-7. 24. Goonewardena P, Dahl N, Ritz#{233}n , van Ommen M GJB, Pettersson U. Molecular Xp deletion in a male: suggestion of a locus for hypogonadotropic hypogonadism distal to the glycerol kinase and adrenal hypoplasia !oci. Clin Genet 1989;35:5-12. 25. Partsch C-J, Sippeli WG. Hypothalamic hypogonadism in congenital adrenal hypoplasia. Horm Metab Res 1989;21:623-5. 26. Maintain F, Cab M, Gain A, Congius R, Arvedi G, Mateddu A, et al. Brief report: deletion of the dystrophin muscle-promoter region associated with X-linked cardiomyopathy. N Engi J Med 1993;329:921-5. 27. Cole TG. Glycerol blanking in triglyceride assays: is it necessary? [Editorial] Clin Chem 1990;36:1267-8. 28 K!otzsch SG, McNamara JR. Triglyceride measurements: a review of methods and interferences. Clin Chem 1990;36:1605-13. 29. Jessen RH, Dass CJ, Eckfeldt JH. Do enzymatic analyses of serum triglycerides really need blanking for free glycerol? Clin Chem 1990;36:1372-5. 30. Desmaze C, Prieur M, Amblard F, Aikem M, LeDeist F, Demczuk 5, et al. Physical mapping by FISH of the DiGeorge critical region (DGCR): involvement of the region in fRmiliRl cases. Am J Hum Genet 1993;53: 1239-49. 31. Borresen AL, Hellerud C, Mller P, S#{248}vik Berg K. Prenatal 0, diagnosis of glycero!-kinase deficiency associated with a DNA deletion on the short arm of the X-chromosome. Clin Genet 1987;32:254-9. Biochem 1984;62:43-50. 15. Seltzer WK, McCabe netic properties of soluble

1. Greenberg F. 1993;9(3):5-10.

Contiguous

gene

syndromes.

Growth

Genet

Horm

5. Kohlschutter A, Willig HP, Schiamp D, Kruse K, McCabe ERB, 3chfifer HJ, et al. Infantile glycerol kinase deficiency-a condition equiring prompt identification. Clinical, biochemical, and mor,hological findings in two cases. Eur J Pediatr 1987;146:575-81. 5. Bartley JA, Patil S, Davenport S, Goldstein D, Pickens J. Duchenne muscular dystrophy, glycerol kinase deficiency, and sdrenal insufficiency associated with Xp21 interstitial deletion. J Eediatr 1986;108:189-92. 1. McCabe ERB. Disorders of glycerol metabolism. In: Scriver CR, 3eaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited fisease, 6th ed. New York: McGraw-Hill, 1989:945-61. 5. Winter JSD, Faiman C. Serum gonadotropin concentrations in igonadal children and adults. J Clin Endocrinol Metab 1972;35:
561-4.

5. Pillers

DM,

powell

BR, McCabe

Towbin JA, Chamberlain JS, Wu D, Ranier J, ERB. Deletion mapping of Aland Island eye

CUNICAL

CHEMISTRY,

Vol. 40, No. 11,

1994

2103