2002, Volume 13, Number 3
David F. Keren, M.D.
QUAD Test Update
A little over a year ago, we began performing the QUAD test for prenatal screening. The Quad Test studies the mother's blood sample for four analytes: alpha fetoprotein (AFP), beta-human chorionic gonadotropin (hCG), unconjugated estriol (uE3), and dimeric inhibin A (DIA). These are used together with information about maternal age, race, weight and diabetes status to estimate her risk for carrying a child with Down syndrome, Trisomy 18 or an open neural tube defect [1-3]. As discussed below, Warde Medical Laboratory is now able to estimate the risk for Smith-Lemli-Optiz Syndrome (SLOS) as well.
The triple test uses only AFP, hCG and uE3. Unfortunately, these three analytes change considerably during the optimal weeks for prenatal testing (weeks 16-18). As a result, even relatively small errors in gestational age increase the chance of false positive and false negative results. The addition of the new marker, dimeric inhibin A (DIA) is important because DIA does not change significantly in concentration during this time period. This consistency helps to dampen the problem of dating that affects the other analytes more severely. DIA also adds to the sensitivity of picking up pregnancies affected with Down syndrome. Because of these factors, last year we recommended replacing the older Triple test with our QUAD test.
In the past six months 85.5% of our samples sent for multiple marker prenatal screening have been for the QUAD test. We compared the numbers of initial positives (an indicator of false positives) for orders of the QUAD test versus the older Triple test for the last six months of 2001.
As the literature predicted, we found a higher false positive rate of screening tests for Down syndrome (8.3%) using the less specific Triple test compared to the QUAD test (7.5%) over this most recent period. With the approximately 14,000 specimens that we estimate will be processed in 2002, the use of the QUAD test over the older test will reduce the false positive rate by almost 1%. This translates into 112 women who will not receive false positive reports if all offices use the QUAD test in 2002.
This, in addition to the increased detection rate of true positive cases documented in the literature is why we strongly recommend using the QUAD test for prenatal screening at this time.
Smith-Lemli-Optiz Syndrome (SLOS)
In addition to this follow-up on the use of the QUAD test, Warde Medical Laboratory has initiated the use of software that detects individuals at risk for carrying a fetus with SLOS as part of our routine QUAD prenatal screening test. SLOS is an autosomal recessive condition associated with several malformations and mental retardation and has an incidence of from 1:20,000 to 1:40,000 . Diagnosis of this syndrome is useful both in prenatal planning and in potential therapy.
SLOS is caused by a mutation in the 3 beta-hydroxysteroid delta (7)-reductase gene (delta 7 reductase). This causes impaired enzymatic conversion of 7-dehydrocholesterol to cholesterol and this results in severe cholesterol deficiency in the fetus . Sera from patients with SLOS have hypocholesterolemia, markedly elevated levels of the precursor, 7-dehydrocholesterol, low testosterone, and low unconjugated estriol in the second and third trimesters of pregnancy . This condition was first recognized in 1964 and was initially called RSH for the surnames of the first three individuals who had this condition. The initials RSH are still used in the title of some articles about SLOS .
SLOS Clinical Features
Children who have SLOS have a wide variety of clinical features.Some are so severely affected that they suffer fetal demise. There have, however, been rare reports of milder forms of the mutation in delta 7 reductase gene that result in a partial, milder version of SLOS. Some of these individuals have only mildly decreased or even low normal levels of cholesterol in the serum . Because of these mild cases, screening for just serum cholesterol in suspected individuals is inadequate to rule out the presence of SLOS. Patients with clinical features of SLOS who have negative biochemical testing for 7-dehydrocholesterol may benefit from studies of 7-dehydrocholesterol in cells cultured in the absence of cholesterol .
All of the affected patients have mental retardation and more than 90% have microcephaly. Facial characteristics in more than half of the patients include one or more of the following: inner epicanthal folds, ptosis, low set abnormal pinnae, cleft palate, small tongue, small mouth, small lower jaw, and broad maxillary alveolar ridges (Table 1).
Table 1: Facial Characteristics of SLOS
Other key features present in more than half of affected patients include: hypotonia, syndactyly of toes (98% of cases have syndactyly of toes 2/3), postnatal growth retardation (that may be affected by diet--see below), and in males hypospadius, bifid scrotum and cryptorchidism . Normal-appearing female genitalia and even persistence of Müllerlian structures may be seen in 46, XY males affected by SLOS  (Table 2).
Table 2: Nonfacial Clinical Features of SLOS
The easily recognized genital malformations in males may explain the predominance of males among those reported to have SLOS. Recent studies suggest that when biochemical means are used rather than physical characteristics for the initial ascertainment of cases, the sex ratio is close to one . Less common features found in these patients include: photosensitivity, Hirschsprung disease, sexual ambiguity, cataracts, polydactyly, cardiac defects, adrenal hyperplasia, lung defects, cerebellar hypoplasia, abnormal cerebral gyri, defects of the liver and kidneys, pyloric stenosis and prenatal growth retardation. The mental function varies widely. Most affected children are moderate to severely retarded; however, some cases have developmental quotients in the mildly retarded range .
Prenatal Detection of SLOS
Palomaki et al. recently published a model for detecting SLOS as part of current Down syndrome screening programs . It can be used in Down syndrome screening programs such as our QUAD or Triple tests that include measurement of unconjugated estriol (uE3). Studies of affected pregnancies have reported uE3 multiple of the median (MOM) levels of from undetectable to 0.65 MOM .
Decreased uE3 levels may also have other causes of clinical significance in prenatal screening. Low levels of uE3 are found in pregnancies with a Trisomy 18 fetus. Since Warde Medical Laboratory has been screening for Trisomy 18 for several years, some cases of SLOS have been detected in this manner.
If an amniocentesis is performed on a woman who was found to be at increased risk for Trisomy 18 by the QUAD test (which would include low uE3 levels) but the cytogenetic study does not demonstrate Trisomy 18, SLOS should be considered in the differential diagnosis. SLOS, however, is not the only possible condition in that situation. In some cases extremely low uE3 values may indicate fetal demise. Alternatively, very low uE3 levels have been reported in serum from patients carrying pregnancies affected by steroid sulfatase deficiency and X-linked ichthyosis . The detection of steroid sulfatase deficiency is useful information for the obstetrician because pregnancies associated with steroid sulfatase deficiency may have prolonged labor.
The levels of AFP and hCG may be normal or decreased in affected pregnancies .By using a cut-off of a 1 in 50 risk, we can detect about 60% of pregnancies carrying an SLOS-affected fetus. With this cut-off, a false positive report is expected to occur in just under 1% of unaffected pregnancies.
Once a prenatal screening test like the QUAD test indicates an increased risk for SLOS, an ultrasound evaluation is helpful both to confirm the gestational age, and also to demonstrate anomalies that often may be seen in affected fetuses. However, a negative ultrasound for anomalies does not rule out SLOS. The diagnostic test for SLOS in this situation is testing of the amniotic fluid sample for the presence of markedly elevated levels of 7-dehydrocholesterol. Typically, the level of 7-dehydrocholesterol is elevated 1000 times or more in the amniotic fluid of affected pregnancies. Warde Medical Laboratory will help your office arrange for this testing by the Kennedy Krieger Institute in Baltimore, Maryland.
SLOS Postnatal Studies
Prenatal screening cannot detect all cases of SLOS. As mentioned above, the cut-off we use of a risk of 1:50 detects approximately 60% of cases. Postnatal diagnosis involves the use of both clinical features and biochemical studies to detect the affected children. Because of the delta 7 reductase deficiency, one might expect the total serum cholesterol and the low-density lipoprotein levels to be low. They usually are. However, normal levels do not rule out SLOS because a small number of cases will have low normal range levels of these lipids.
Biochemical demonstration of markedly elevated levels of 7-dehydrocholesterol with low serum cholesterol identifies the patients with SLOS. The reader should note that 7-dehydrocholesterol is found normally in very low amounts in the serum of controls. In SLOS these levels are markedly elevated. Since some clinical features of SLOS resemble adrenal insufficiency, demonstration of normal serum cortisol, ACTH and electrolytes can help to rule out that abnormality.
In a few cases, atypical cases with clinical features of SLOS may have normal levels of 7-dehydrocholesterol. For these individuals, a skin biopsy to provide cultured fibroblasts for enzymatic testing may be needed to provide a definitive diagnosis. Because the vast majority of cases have both clinical features and markedly elevated levels of 7-dehydrocholesterol, skin biopsy is needed only rarely.
Unfortunately, at the present time, diagnostic tests for the specific mutations in the delta 7 reductase gene is not recommended. This reflects the large number of mutations that have been found in the literature. However, in families where specific mutations have already been detected, this information may be used for early diagnostic procedures such as chorionic villous sampling that are performed during the first trimester of pregnancy.
A variety of radiological studies may be performed to demonstrate abnormalities seen in SLOS. Gastrointestinal manifestations are common features of SLOS. Therefore, studies to detect pyloric stenosis such as a barium swallow or abdominal ultrasound may be fruitful. Hirshprung disease may be detected by a standard barium enema, or may even be detected by x-rays of the abdomen. A rectal biopsy is useful to provide the confirmation of the radiological findings. MRI of the brain may reveal cerebellar hypoplasia and/or abnormal cerebral gyri. Numerous heart defects can be detected by ECHO studies together with EKG. Ultrasound studies may also be useful in confirming the presence of genitourinary and renal abnormalities.
Other specialty studies that are important in the postnatal evaluation include ophthalmologic studies to detect cataracts, hearing tests, psychological and intelligence quotient studies to detect mental retardation.
Therapy for SLOS patients
Because of the photosensitivity, the patients must avoid excessive sun exposure. Generous use of sunscreen is recommended. Diagnosis of SLOS is helpful both in prenatal planning and in therapy. Since the genetic defect interrupts normal sterol biosynthesis many functions of the body are affected, including development of the brain, cell membranes, pathways for bile acid synthesis and other steroid hormone synthesis. Infants require a high level of cholesterol and breast milk alone only provide about half of the needed amount. Since SLOS infants cannot effectively synthesize cholesterol, and diarrhea due to intestinal absorption problems often exists in these patients, supplemental therapy is needed. Treatment of SLOS includes a high cholesterol diet sometimes with supplements of bile acids. The dietary supplements have considerably helped the growth of some affected children and decreased their problems with photosensitivity [6, 11]. Dietary supplements have also resulted in anecdotal reports of improved mood and mentation.
Unfortunately, the response to cholesterol supplementation is not uniform with results varying from considerable improvement to more modest benefits. It is hoped that cholesterol supplementation will also decrease the levels of 7-dehydrocholesterol by feedback inhibition of hydroxymethylglutaryl coenzyme A reductase. However, though clinical improvement has been demonstrated, consistent reduction in 7-dehydrocholesterol levels has yet to be confirmed. Recently, some studies have added hydroxymethylglutaryl coenzyme A reductase inhibitors to see if use of statins may improve the outcome.
The success, albeit modest in some cases, in the postnatal cholesterol supplementation trials have raised the issue of possible prenatal, or fetal supplementation. Irons et al. have demonstrated that by use of fetal intravenous and intraperitoneal transfusions of fresh frozen plasma that the level of fetal cholesterol measured in cordocentesis samples increased . In addition, they demonstrated an increase in fetal erythrocyte cell mean corpuscular volume. These findings add some urgency to the early detection of SLOS. Whereas prenatal supplementation cannot cure the enzyme defect, it may be able to ameliorate some of the congenital effects.
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