Defense Date


Document Type


Degree Name

Doctor of Philosophy


Human Genetics

First Advisor

William B. Rizzo


Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder due to deficiency of the fatty aldehyde dehydrogenase (FALDH) component of fatty alcohol:NAD+ oxidoreductase (FAO). We investigated the enzymatic defect in SLS in order to elucidate the role of FALDH in fatty aldehyde and fatty alcohol metabolism.

Genetic studies were performed to investigate carrier detection for SLS. Cultured skin fibroblasts from normal controls, SLS obligate heterozygotes, and SLS homozygotes were assayed for FAO and FALDH activities using 18-carbon substrates. In SLS homozygotes, mean FAO and FALDH activities were 8% of normal, and there was no overlap between the homozygote and heterozygote ranges. We demonstrated that mean FAO activity in SLS obligate heterozygotes was 78 ± 16% of normal (mean ± SD); whereas mean FALDH activity was 60 ± 15% of normal. Using both FAO and FALDH assays in concert, none of the SL8 obligate heterozygotes (n=11) had both FAO and FALDH activities which overlapped the normal range (n=12). Therefore, we conclude that measurement of FAO and FALDH activities in cultured skin fibroblasts using 18-carbon substrates is useful for SLS carrier detection.

Prenatal diagnosis was undertaken to monitor FAO and FALDH activities in four pregnancies at-risk for SLS. Enzymatic results in one case using cultured amniocytes obtained during the second trimester predicted an affected SLS fetus, which was confirmed at termination of the pregnancy. Another at-risk fetus was predicted to be affected with SLS using cultured chorionic villi cells obtained during the first trimester, and fetal skin fibroblasts obtained after termination of the pregnancy confirmed a profound deficiency of FAO and FALDH activities. Two other fetuses were correctly predicted to be unaffected. These results demonstrate that SLS can be diagnosed prenatally during either the first or second trimester of pregnancy using enzymatic methods.

In order to better understand the role of FALDH in fatty alcohol oxidation, we determined the subcellular localization of FALDH in human liver, a human cultured hepatocyte cell line (HepG2), and human cultured skin fibroblasts. Differential centrifugation and density gradient centrifugation in Nycodenz were utilized to separate subcellular organelles. Organelle-specific enzyme markers confirmed the subcellular separations that were attained. FALDH activity was primarily localized to the microsomes in human liver, a cultured HepG2 cells, and human cultured skin fibroblasts.

FALDH was solubilized from human liver microsomes and purified 167-fold by column chromatography. Purified FALDH had a subunit molecular weight estimated by SDS-PAGE to be 54,000 daltons. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of purified FALDH indicated that the enzyme formed large, polymeric protein aggregates with a molecular weight greater than 500,000 daltons. FALDH was NAD+-dependent, had optimal catalytic activity at pH 9.8, and was thermolabile at 47°C. Km and Vmax values were determined for saturated and unsaturated aliphatic aldehydes ranging from 2 to 24 carbons in length, as well as dihydrophytal, a 20-carbon branched chain aldehyde. FALDH was active towards long-chain fatty aldehydes greater than 6 carbons in length. FALDH was sensitive to inhibition by disulfiram, iodoacetamide, iodoacetate, a,p-dibromoacetophenone, and p-chloromercuribenzoate, but it was unaffected by magnesium ions. These investigations represent the first purification and characterization of human microsomal FALDH.


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