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Research: Mass Spectrometry

Disorders of steroid biosynthesis and metabolism
The use of mass spectrometry in investigating human disorders of steroid biosynthesis and metabolism has been the underlying theme of this PI’s research since 1967. The topic of most recent interest is developing methodologies for prenatal diagnosis and neonatal screening for rare disorders that affect the production of estriol during pregnancy. Estriol is an estrogen that is largely produced from fetal precursors and its synthesis increases about 1000 fold during gestation. Estriol itself seems to have only minor functions but if produced in low amount can indicate other serious fetal deficiencies. There are several single gene causes of low estriol and we are attempting to develop methods for their diagnosis. The starting point for these studies has been the triple marker screening test given to one half of pregnant women to detect neural tube defects. This test includes estriol, and consequently, occasional low values are found which concerns genetic counselors, physicians and the mothers-to-be. Our program is to diagnose these conditions non-invasively.

Smith-Lemli Optiz syndrome:
The most severe disorder, Smith-Lemli-Opitz syndrome (SLOS), is also one of the most common, affecting up to 1:12000 newborns. It is a severe malformation and mental retardation disorder caused by cholesterol deficiency. The enzyme 7-dehydrosterol-7-reductase (Dhcr7), responsible for catalyzing the final step in cholesterol biosynthesis, is inactive resulting in cholesterol deficiency.

This disorder has been diagnosed prenatally by the finding of elevated levels of 7- and 8-dehydrocholesterol in amniotic fluid. This invasive test has only been carried out on patients who are at risk for the disorder through having a previously affected child. Dr Shackleton reasoned that maternal urine and serum must also contain markers for the disorder and if we could find them it would allow non-invasive testing, and the possibility of screening for SLOS.

After obtaining samples from women with a SLOS fetus we searched for putative steroids that had an extra double bond. We found several and chose two that we thought would be the most useful for analysis. One of them was 8-dehydroestriol whose structure is shown below in comparison to the conventional estriol produced by all pregnant women carrying a normal fetus. A collaboration was started with synthetic chemists at Rice University and compounds identical to our analytes were synthesized, thereby proving our putative structures. This discovery has been patented. We measured the specific analytes in many more urine and serum samples of SLOS pregnancies and proved that we had the basis for a viable biochemical test. Our finding has led to us being included in a multi-center project, which includes the State of California, to test the feasibility of prenatal screening for SLOS. Over four years we will measure our analytes in a low-estriol group chosen from one million pregnant women. This study will not only prove the efficacy of our methodology but should also give a better figure for the incidence of SLOS.

Our SLOS studies are not restricted to prenatal testing. We are currently studying 35 patients with the disorder (in association with Dr. Forbes Porter of NICHD) and are measuring adrenal steroids and their metabolites to ascertain whether individuals have potential adrenal insufficiency secondary to cholesterol deficiency.
We are collaborating with Dr Porter on studies of a mouse model for SLOS. Our main focus is on formation of neurosteroids, important compounds made in the brain, which primarily modulate the action of membrane receptors such as NMDA and GABAA. SLOS is a mental retardation disorder associated with severe behavioral problems such as cognitive delay, sensory hyperactivity, self-injurious behavior, destruction, sleep deprivation and autism. Certain neurosteroids, e.g., allopregnanolone are anxiolytic, anesthetic, improve memory, and reduce stress, and reduced levels, as potentially found in SLOS may have behavioral impact. We believe that the improved behavior of SLOS patients treated with cholesterol may act through the neuroactive steroid pathway since the blood brain barrier is impervious to cholesterol itself. We have found novel neuroactive steroids of distinctive structure in urine from SLOS patients and we are now beginning a study on neurosteroid synthesis in the brain of the mouse model for the disorder. These studies in the long-term may lead to new therapies for the disorder.

It is possible that, in the future, gene therapy with the Dhcr7 gene could be used antenataly to increase cholesterol synthesis and alleviate some of the most severe aspects of the condition. As a first step to investigating this, Dr Shackleton and Dr Watson propose to employ gene therapy in mouse models of the disorder. With the collaboration of Dr Porter we have observedbreeding animals and establish a colony at our institution (CHORI). Dr Porter has currently three models: a Dhcr7 null mutation with deleted exons 3-5, which die on first day of life, and two missense mutations, T93M and L99P, which survive but still exhibit SLOS characteristics. The less severe missense mutants will be used in initial experiments to develop gene therapy vectors and protocols, while the null mutant will ultimately be used for demonstration of efficacy in prenatal therapy. The principle of gene therapy is to provide the missing gene function and thus restore metabolic balance. Adeno-associated virus (AAV) vectors are proposed to deliver Dhcr7 DNA to cells of the mutant mice. Primarily liver cells will be targeted by intravenous administration of vector, while brain will be targeted by intrathecal (into the cerebrospinal fluid) administration. We will determine the success of the therapy by measuring the degree to which cholesterol synthesis has been increased. This can be determined by measuring circulating cholesterol and relating the concentration to that of Dhcr7 -/- animals that have not received the therapy. It is likely that efficacy can also be measured by determining the 7-DHC/C ratio in serum and tissues as well as by analysis of steroids derived from these precursors. In human SLOS urinary steroids are particularly effective analytes for both diagnosis and assessing disorder severity. We believe this could also be the case in mice. Measuring the ratio of excreted conventional to 7-dehydrosteroids may offer a non-invasive, non-sacrificial way of assessing the success of the therapy, and this ratio could be monitored sequentially from newborn to adulthood. Shackleton and Watson are currently preparing a proposal for submission to the NIH on this subject.

Antley-Bixler Syndrome:
A second malformation syndrome, Antley-Bixler syndrome, has recently become a major focus of research. This disorder is associated with craniofacial and other skeletal deformities, as well as genital ambiguity. It had been noted that patients with the disorder frequently had a steroid synthesis pattern indicative of partial 17- and 21-hydroxylase deficiencies.

We have recently been studying several patients with this and a related disorder in association with Dr. Wiebke Arlt in Birmingham, England and colleagues in Japan, Poland and Germany. We expected that the disorder was associated with deficiency of a redox partner for hydroxylase enzymes since no mutations had been found in the 21- and 17-hydroxylase genes. In fact, it was extremely unlikely that mutations in more than one hydroxylase enzyme could exist. We obtained DNA samples from two patients with the disorder and did sequence analysis of the gene encoding P450 oxidoreductase, the enzyme important for electron transfer from NADPH to P450C17 and P450C21. We found four mutations, all patients being compound heterozygous. These data have recently been published in the Lancet (363:2128-2135). Recently we have diagnosed another six patients with the disorder and are currently completing gene sequencing. In addition to discovering the cause of the disorder, we have been researching biochemical diagnostic techniques. In particular, we followed a pregnancy carrying an affected fetus and believe that we are able to diagnose the condition prior to mid-gestation.

Other studies:
Our other major studies have focused on the enzyme 11ß-hydroxysteroid dehydrogenase (11ßHSD), a subject Dr Shackleton has been involved with for 30 years. This steroid metabolic enzyme has importance for maintaining a correct cortisol/cortisone balance in multiple organs. Since these studies began we’ve shown that two enzymes (11ßHSD 1 and 11ßHSD 2) exist with very different responsibilities. Current studies involve the relevance of theses enzymes to hypertension, diabetes, obesity and the eye and are being conducted in collaboration with Prof Paul Stewart of the University of Birmingham, England.

Multiple other small collaborative projects are continually being tackled, the majority regarding diagnosis of rare forms of steroid hypertension. For some years we were a center for the mass spectrometric characterization of variant hemoglobins and this is still undertaken at a limited level.

Available mass spectrometry equipment at CHORI:
Shackleton Lab: Hewlett-Packard 5971 GC/MS; Thermofinnigan Polaris ion-trap GC/MS; Micromass BioQ LC/MS/MS

Ames, Shiginaga Lab: Hewlett-Packard Engine CI GC/MS; Micromass Quattro LC/MS/MS; Bruker Autoflex


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