Clinical Mass Spectrometry Laboratory
Kennedy Krieger Institute
707 North Broadway
Baltimore, Maryland 21205
The discovery that patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) have a fundamental defect of cholesterol biosynthesis manifest as high levels of 7-dehydrocholesterol has provided geneticists with a simple biochemical test for prenatal diagnosis of RSH/SLOS. In all knows affected RSH/SLOS pregnancies tested to date, the level of 7-dehydrocholesterol, the immediate precursor of cholesterol, has been markedly increased in mid-trimester amniotic fluid. Similarly, cultured amniocytes and chorionic villus from affected pregnancies have abnormally increased ratios of 7-dehydrocholesterol to cholesterol. Since 1993, more than 150 pregnancies at risk for RSH/SLOS have been tested by one of several biochemical measures and the outcomes correctly predicted. In addition, because an abnormal triple screen, and in particular a low estriol level, is found in most RSH/SLOS pregnancies, measurement of the amniotic fluid 7–dehydrocholesterol level has also been used to evaluate unexplained low maternal serum estriol levels.
Notwithstanding the relative technical simplicity and high accuracy of prenatal testing for RSH/SLOS, there are several important considerations and steps to be taken when planning to test a pregnancy for RSH/SLOS. Accordingly, we have prepared the following information and procedures for prenatal diagnosis of RSH/SLOS.
Because of the need for careful planning and laboratory coordination for prenatal studies, the Kennedy Krieger Institute Clinical Mass Spectrometry Laboratory should be contacted before or as soon as possible after the pregnancy has been established to discuss the specific case and various options for prenatal diagnosis and monitoring of the pregnancy. The prospective parents should be counseled in detail about the specific diagnostic procedures and the biochemical testing that may be undertaken as early as possible so informed diagnostic choices can be made in a timely manner.
Diagnosis of Propositus
Prior to prenatal testing, the diagnosis of an abnormally increased level of 7-dehydrocholesterol in the index patient must be established with certainty because, historically, about 20% of patients who carry a clinical diagnosis of RSH/SLOS have not had the characteristic sterol abnormality now equated with the diagnosis. If the biochemical diagnosis of RSH/SLOS was not made in this laboratory, the results of testing done elsewhere or a repeat plasma sample should be submitted. If the index patient is not available for testing, the following should be sought as possible sources of test material:
A Guthrie blood card may not be reliable for biochemical diagnosis of RSH/SLOS if stored at room temperature for more than a year, but the card should nevertheless be submitted for analysis if it is the only sample remaining. (Unfortunately, some state screening programs discard Guthrie cards after only 6 months.) Plasma samples from non-routine tests, such as special endocrine studies, are often stored for many years at the reference laboratory. If no specimen for biochemical testing can be found, detailed clinical records should be forwarded to Dr. Kelley for evaluation and consultation with the referring geneticist or obstetrician to determine the likelihood that the patient had the classical biochemical abnormality. Carrier status in one or both parents also can be established by measurement of 7-dehydrocholesterol levels in cultured fibroblasts or transformed lymphoblasts. However, carrier testing takes at least 6 weeks to complete and may be negative in up to 5% of obligate RSH/SLOS heterozygotes. Thus, unless knowledge of carrier status is important for their reproductive planning, most parents elect to have the more reliable prenatal diagnosis without prior carrier testing. 7-dehydrocholesterol Since the gene for RSH/SLOS has recently been found and mutations characterized, it is possible that carrier testing by DNA analysis may become available in this or another laboratory sometime in 1999. Although we do not require absolute certainly of a prior biochemical diagnosis before undertaking prenatal testing, every effort should be made to make a biochemical diagnosis, because prenatal testing without a confirmed diagnosis of RSH/SLOS reduces the chance of identifying a recurrence of the propositus’s disorder.
If arrangements for amniocentesis are made, measurement of maternal serum levels of chorionic gonadotropin, alpha-fetoprotein, and unconjugated estriol should be scheduled for 15-16 weeks gestation, and a sonographic study for gestational dating should be performed per routine. At amniocentesis, sufficient fluid should be removed to provide the following samples:
In addition, the following control specimens are requested:
Samples other than the back-up amniocyte cultures and remaining spun amniotic fluid should be shipped to the Clinical Mass Spectrometry Laboratory. Chromosome analysis and other prenatal testing, as indicated, should be performed by the referring laboratory per routine.
Chorionic Villus Sampling
Because the level of 7-dehydrocholesterol has been found to be increased in all fetal tissues including the placenta, it is likely that RSH/SLOS can be diagnosed as accurately by measurement of 7-dehydrocholesterol in chorionic villus obtained during the first trimester as by amniotic fluid testing. However, because experience with prenatal diagnosis of RSH/SLOS by direct analysis of chorionic villus is still somewhat limited (only three positive CVS tests as of July, 1997), the need for a follow-up amniotic fluid study because of an ambiguous result with CVS testing cannot be excluded. For RSH/SLOS prenatal testing by CVS, the following samples should be obtained.
In the unlikely event of an ambiguous result, 2 T25 not-at-risk CVS cultures will be requested in addition to the patientęs CVS cells.
In addition to direct analysis of villus material, assay of villus cells after 1-2 weeks of growth in lipid-depleted culture medium may be required if results of the direct analysis are non-diagnostic. Chromosome analysis and other prenatal testing should be performed by the referring laboratory as indicated.
Targeted sonography for fetal sex, fetal anomalies, and growth parameters should be considered at the time of amniocentesis, especially when there is uncertainty about the biochemical diagnosis of the propositus. Special attention should be directed toward identifying abnormal growth, microcephaly, cleft palate, cardiac defects, renal anomalies, genital anomalies, polydactyly, and 2/3 toe syndactyly.
Results on amniotic fluid and direct CVS analysis are available within 2 or 3 days. Studies of cultured amniocytes or villus cells require 2 to 3 weeks to complete. In the event of an abnormal result and a decision to terminate the pregnancy, further information will be provided about procedures for biochemical confirmation of the diagnosis in fetal material. We also request confirmation of the normal outcome of pregnancies with normal test results.
All specimens for analysis should be shipped to:
Amniotic fluids and villus samples for direct analysis should be sent frozen on dry ice. Tissue culture flasks (T25) should be filled with medium and shipped at room temperature in a well-insulated styrofoam box to protect the cells from extreme temperature changes. All samples should be shipped by overnight express carrier to arrive on a weekday.
To assure correct and expedited handling of diagnostic samples, the Clinical Mass Spectrometry Laboratory should be notified of the anticipated testing date and also on the day of shipment of any samples. Failure to notify the laboratory may delay sample processing and reporting of results. Any questions about samples or procedures can be directed to Dr. Kelley of Dr. Kratz.
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Cunniff C, Kratz LE, Moser A, Natowicz MR, Kelley RI. The Clinical and Biochemical Spectrum of Patients with RSH/Smith-Lemli-Opitz Syndrome and Abnormal Cholesterol Metabolism Am J Med Genet 1997; 68:263-269.
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Tint GS, Irons M, Elias E, Batta AK, Frieden R, Chen TS, Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. New Eng J Med 1994; 330:107-113.