Diagnosis of Smith-Lemli-Optiz syndrome

Details Diagnosis of Smith-Lemli-Optiz syndrome Diagnosis of Smith-Lemli-Optiz syndrome

2002-02-15

2004-10-26

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Glycoproteins

C436S173000, C436S177000, C436S811000, C436S071000, C436S139000, C436S817000

Reexamination Certificate

active

06808932

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of diagnosis of hereditary conditions such as Smith-Lemli-Optiz syndrome, particularly prenatal diagnosis.
BACKGROUND OF THE INVENTION
Smith-Lemli-Optiz/RSH syndrome (SLOS) is a genetic disorder that affects the development of children both before and after birth SLOS affects about 1:20,000 individuals. The syndrome was first described in 1964 in three boys with poor growth, developmental delay, and a common pattern of congenital malformations including cleft palate, genital malformations, and polydactyly (extra fingers and toes). In 1993 scientists discovered that children with SLOS are unable to make sufficient cholesterol.
The Smith-Lemli-Opitz syndrome (SLOS) is caused by impaired activity of the enzyme 3&bgr;-hydroxsterol, &Dgr;
7
-reductase (7DHCR)(Irons et al.,
Lancet
341: 1414, 1993; Tint et al.,
N Engl J Med
330: 107-113, 1994), which is involved in the enzymatic conversion of 7-dehydrocholesterol to cholesterol in one of two proposed routes of cholesterol biosynthesis (Scheme 1). The defect in 7DHCR results in an abnormal accumulation of 7- and 8-dehydrocholesterol (1 and 2). More than 60 enzyme mutations have been detected in SLOS-affected individuals (Fitzky et al.,
Proc Natl Acad Sci USA
95; 8181-8186, 1998; Moebius et al.,
Proc Natl Acad Sci USA
95: 1899-1902, 1998; Wassif et al.,
Am J Hum Genet
63: 329-338, 1998).
The challenge in prenatal diagnosis has been the identification of a non-invasive test that involves detection of definitive and SLOS-specific analyte(s), and which can be performed early in pregnancy. Many of the conventional SLOS screening assays involve detecting varying levels of 7-DHC, cholesterol or estriol (E
3
) or a combination thereof, in chorionic villus (CV) or amniotic fluid samples, each of which are invasive tests and can pose a risk to the fetus. TABLE 1 lists the most common conventional SLOS markers for the screening of SLOS affected fetuses.
Table 1: Exemplary SLOS Markers
Low Cholesterol levels in CV biopsies and amniotic fluid.
Increased 7-DHC levels (Dehydrocholesterol) in CV and amniotic fluid.
Increased 8-DHC levels (dehydrocholesterol II) in CV and amniotic fluid.
Low unconjugated estriol levels in serum (non-specific marker)
Since fetal cholesterol is a precursor to estriol (E
3
), E
3
is decreased in SLOS affected pregnancies (Donnai et al.,
J Med Genet
23: 6471, 1986; McKeever and Young,
J Med Genet
27: 465466, 1990; Abuelo et al.,
Am J Med Genet
56: 281-285, 1995; Rossiter et al.,
Am J Med Genet
56: 272-275, 1995). However, detection of E
3
levels is not specific for SLOS-affected pregnancies. Currently, unconjugated scrum estriol (uE
3
) is measured in about 50% of all United States pregnancies as part of the “triple marker screening” for chromosomal aneuploidies and neural tube defects (Palomaki et al.,
Am J Med Genet
176: 1046-1051, 1997). In 1999, Bradley and co-workers (Bradley et al.,
Amer. J. Med. Gen.
82:355-358, 1999) published a retrospective study of 26 SLOS pregnancies in which uE
3
in serum had been measured and determined that the mean uE
3
level was 0.23 of normal median (multiples of the median, MOM). Assaying for low estriol levels in maternal serum MsuE
3
along with sonography have also been suggested for diagnosing RSHISLOS (Kratz, L. E., Kelley, R. I.,
Amer. J. Med. Gen.
82:376-381, 1999) as well as identifying patients with low maternal urinary levels of estriol (McKeever and Young, 1990) at mid-gestation.
Many SLOS diagnostic methods have been suggested in which the level of 7-DHC is detected due to the increase of this cholesterol precursor in SLOS affected patients. High levels of 7-DHC and 8-DHC have been detected in amniotic fluid and in CV biopsies of SLOS patients (Rossiter, J. P. et al.,
Amer. J. Med. Gen.
56:272-275, 1995; Tint, G. S. et. al.,
Prenat. Diagn.
18:651-658, 1998; Irons, M. B., Tint, G. S.,
Prenat. Diagn.
18:369-372, 1998; Kratz, L. E., Kelley, R. I.,
Amer. J. Med. Gen.
82:376-381, 1999). High levels of 7-DHC have also been detected in CV biopsies of SLOS patients as early as the first trimester (Sharp, P. et al.,
Prenat. Diagn.,
17(4): 355-361, 1997). In addition to detection of 7-DHC and 8-DHC, high levels of lathosterol (cholest-7-en-3beta-ol), a 7-DHC precursor, have also been detected in amniotic fluid.
Mills, K. et al.,
Pediatric Research
39(5): 816-819 (1996) describe a method for detecting SLOS by determining the ratio of 7-DHC (a cholesterol precursor) to cholesterol in chorionic villus (CV) samples. Mills et al. determined that cholesterol synthesis via 7-DHC occurs in the placenta and/or fetus at 10 weeks of gestation and that prenatal diagnosis by CV biopsy is possible. While this test can detect SLOS early in gestation, CV biopsy is an invasive procedure and is associated with some risk to the fetus and patient.
Recently, it was shown that mid-gestational urine from a SLOS affected pregnancy contains metabolites unsaturated analogs of the compounds estriol (E
3
) and pregnanetriol (PT). These compounds were suggested to be synthesized or metabolized from fetal 7- or 8-DHC (Shackleton et al.,
Steroids.
1999a, 64(7): 446-52; Shackleton et al.,
J. Clin. Endocrinol. Metab.
1999b, 84(3): 1157-9; Shackleton et al.,
Steroids.
1999c, 64(7): 481-90).
Shackleton et al 1999a, disclose that either 7-DHPT or 8-DIPT of the 3,16,20 and 3,17,20 (triol structures) series was present in the maternal urine of one healthy 35 year-old women carrying a SLOS fetus at 17 weeks gestation. The authors provisionally characterize the SLOS metabolite as 5&bgr;-pregn-7(or 8-)-ene-3&agr;,17&agr;,20&agr;-triol; 5&bgr;-pregn-7(or 8-)-ene-3&agr;,16&agr;,20&agr;-triol; 5&agr;-pregn-7(or 8-)-ene-3&agr;, 16&agr;,20&agr;-triol; 5&agr;-pregn-7(or 8-)-ene-3&agr;,17&agr;,20&agr;-triol and/or 5&agr;-pregn-7(or 8-)-ene-3&bgr;,16&agr;,20&agr;-triol. The authors indicated that the major SLOS metabolite compound is either 50pregn-7-ene-3&agr;,17&agr;,20&agr;-triol (7-DHPT) and/or 5&bgr;-pregn-8-ene-3&agr;,17&agr;,20&agr;-triol (8-DHPT). The authors did not indicate if the tentative SLOS metabolite was a mixture of the two epimers or pure 7-DHPT or 8-DHPT and no isolation of the specific compound was attempted. Furthermore, Shackleton et al. 1999a did not show detection of the analytes prior to 17 weeks gestation.
In a separate study of three young infants affected with SLOS, the authors detected a SLOS metabolite(s) and provisionally identified the compound(s) as 3&bgr;,16&agr;-dihydroxy-5,7-pregnadien-20-one; 3&bgr;,16&agr;-dihydroxy-5,8 (or 9-)pregnadien-20-one; homologues of 16&agr;-hydroxy-DHEA, as well as the7- or 8- epimer of 5&bgr;-pregnene-3&agr;,17&agr;,20&agr;-triol (Shackleton et al.,
Steroids.
1999c, 64(7). 481-90). In short, while these studies narrow the possibilities of the identity of a unique SLOS analyte, they failed to confirm the identification due to the complexity of the mass spectra profile of the biological sample and the lack of appropriate reference compounds.
While these two SLOS specific metabolites were tentatively characterized by Shackleton et al., the actual structures and identification of the two SLOS specific analytes (&Dgr;
7
or &Dgr;
8
) was not determined. Depending upon the sensitivity of the detection system, a detectable amount of these SLOS analyte may be found in normal patients, an assay which only detected the presence of a epimer mixture of these compounds or the wrong epimer without proper controls, could lead to a high frequency of false positives and false negatives, making the assay unpredictable, unreliable and not commercially viable. These risks of false positives and false negatives are further exacerbated when one considers that low levels of SLOS analyte levels are found in affected SLOS individuals, thus necessitating the use of sensitive detection methods such as gas chromatography/mass spectroscopy (GC/MS).
Currently, only pregnancies at 25% risk for SLOS are routinely subjected to testing by Dehydrocholesterol (DHC) measurement, with second

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