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Biomedical subjects

D Feldman

Publications and source records attributed to D Feldman.

At least 127 records · Page 7Linked to original sources
PubMed1994

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain.

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease that results from mutations in the gene encoding the vitamin D receptor (VDR). In this study of two siblings showing classical features of HVDRR, cultured dermal fibroblasts were used to characterize their VDR and assess responsiveness to 1,25-dihydroxyvitamin D3 treatment. The VDR displayed normal affinity and binding capacity for [3H]1,25-dihydroxyvitamin D3; however, the cells failed to exhibit induction of 25-hydroxyvitamin D 24-hydroxylase activity when treated with hormone. A decreased affinity of liganded VDR for DNA cellulose suggested that the defect was localized to the DNA-binding domain. Exons 2 and 3 of the VDR gene, which encode the two zinc fingers in the DNA-binding domain, were amplified and sequenced by polymerase chain reaction. Both siblings exhibited a G to A missense mutation (CGG to CAG) in exon 3, which results in the replacement of Arg77 by Gln at the base of the second zinc finger. This mutation has been described previously in two unrelated cases of HVDRR by Sone et al. It is unclear at this time whether these kindreds might be distantly related and, therefore, harbor the same mutation, or whether this represents a mutational hot spot in the VDR gene.

Amino Acid Sequence↗
PubMed1994

ACTH-independent massive bilateral adrenal disease (AIMBAD): a subtype of Cushing's syndrome with major diagnostic and therapeutic implications.

A 49-year-old man with classic manifestations of Cushing's syndrome had undetectable levels of ACTH, lack of suppression of hypercortisolism with dexamethasone in doses of 2, 8, or 16 mg per day, bilaterally enlarged adrenal glands on MRI, and bilateral adrenal uptake of iodocholesterol. Preoperative treatment with ketoconazole lowered blood pressure and serum cortisol and produced symptoms of steroid withdrawal. Bilateral adrenalectomy revealed massively enlarged adrenal glands (left: 199 g, right: 93 g). Sequencing of the gene encoding the stimulatory G protein, GS alpha, did not show either of two activating mutations previously reported in patients with McCune-Albright syndrome or acromegaly. Twenty-three previous cases of Cushing's syndrome due to ACTH-independent massive bilateral adrenal disease (AIMBAD) have been reported. AIMBAD may cause confusion in the differential diagnosis of Cushing's syndrome as endocrine testing suggests a unilateral, ACTH-independent process while adrenal imaging demonstrates bilateral abnormalities. Bilateral adrenalectomy is curative and appears to carry little risk of Nelson's syndrome. The pathogenesis of AIMBAD appears to be heterogeneous, as recent reports have demonstrated GIP-mediated hypercortisolism and familial AIMBAD. Transition from Cushing's disease to ACTH-independence is not supported by the available data. Future cases of AIMBAD should be investigated carefully to further elucidate the pathogenesis of this disorder.

Adrenal Glands↗
PubMed1994

Mitochondrial schwannopathy and peripheral myelinopathy in a rabbit model of dideoxycytidine neurotoxicity.

BACKGROUND: The reverse transcriptase inhibitor, 2',3'-dideoxycytidine (ddC), causes a dose-limiting peripheral neuropathy in humans, the mechanism of which is unknown. Rabbits given ddC develop peripheral myelinopathy and axonopathy, but it has not been determined if either the myelin or axonal changes are primary or if they occur concurrently. EXPERIMENTAL DESIGN: To characterize sequential development of the ddC-induced neuropathy, 40 rabbits were given either vehicle or ddC by oral intubation at a dose of 35 mg/kg per day for 24 weeks. Electrophysiologic studies, pathologic examination of peripheral and central nervous system and skeletal muscle, and biochemical analysis of the sciatic nerve were performed at baseline (electrophysiology only) and after 8, 12, 16, 20, and 24 weeks of treatment. RESULTS: Neuropathologic changes in peripheral nerves were first evident at 16 weeks and were more pronounced at 20 and 24 weeks; onset of paresis occurred at week 20, whereas clear electrophysiologic deficits were seen only at week 24. Electrophysiologic changes were prolonged F-waves (measure of proximal motor conduction) and minor changes in distal conduction measurements. Pathologic changes included myelin splitting, intramyelinic edema, demyelination, and remyelination of the largest diameter nerve fibers in the ventral root and sciatic nerve. Axonal degeneration and reduction in axonal diameter were seen. Enlarged mitochondria with abnormal ultrastructure were present in Schwann cells of those animals with a myelinopathy. Mitochondrial abnormalities or other signs of degeneration were not seen in neurons of the dorsal root ganglia or in skeletal muscle. Significant changes were not present in myelin protein composition, myelin lipid composition, or activity of the myelin-specific enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase. Major reductions in levels of protein zero (P0, the homophilic adhesion protein of myelin) were not seen; however, the turnover rate of P0 was reduced as P0 messenger RNA expression in ddC-treated sciatic nerves decreased to 30 to 50% of control values. CONCLUSIONS: The peripheral neuropathy caused by ddC in rabbits is characterized as a myelinopathy of the proximal portion of the nerve fibers and as an axonopathy involving both proximal and distal fibers. The myelinopathy was associated with enlarged and abnormally shaped mitochondria in Schwann cells and is consistent with an effect of ddC on structure and function of Schwann cell mitochondria. Altered Schwann cell metabolism was evident by reduced levels of P0 messenger RNA, loss of homophilic myelin adhesion at the intraperiod line, and subsequent intramyelinic edema. Because axonal degeneration occurred concurrently with the myelin changes, it could not be determined if axonal changes were secondary to serve myelinic edema or if they represented a primary effect of ddC on neurons.

Animals↗
PubMed1994

Oncocytic mucoepidermoid carcinoma of the parotid gland.

Oncocytic mucoepidermoid carcinoma of the salivary gland is rare. We describe a 60-year-old woman who presented with a slowly growing left parotid mass. The patient underwent a total parotidectomy, and her postoperative course was uneventful. The gland was enlarged and showed a partially cystic mass containing clear mucoid material. Microscopically, the entire mass showed variably sized cysts lined predominantly with oncocytes and a few mucous goblet cells. Histochemical stains for mitochondria, such as phosphotungstic acid-hematoxylin, confirmed the presence of oncocytes. The recognition of this variant is important, since most of the other primary oncocytic lesions of the salivary glands are benign. The tumor in this case is considered to be a low-grade carcinoma; therefore, complete surgical excision and long-term clinical follow-up are adequate management.

Carcinoma, Mucoepidermoid↗
PubMed1993

Cloning and expression of the gene from Candida albicans that encodes a high-affinity corticosteroid-binding protein.

We have previously demonstrated the presence of a corticosteroid-binding protein (CBP) in Candida albicans and speculated on its homology to the glucocorticoid receptor. To explore this relationship further, we cloned the CBP gene. Our strategy employed sequencing enzymatically derived peptide fragments from purified CBP and using this information to synthesize degenerate oligonucleotide primers for use in the PCR. A 117-bp fragment amplified from C. albicans DNA was then used to screen a genomic library. Hybridizing clones were isolated, and DNA sequencing revealed an open reading frame of 1467 bp which encoded a protein with a molecular weight of 55,545. Southern analysis demonstrated that the gene was present at a unique locus within chromosome R of the Candida genome, while Northern analysis showed that the gene was expressed in C. albicans as a 1.8-kb transcript. CBP was over-expressed in Saccharomyces cerevisiae, and it exhibited an apparent dissociation constant (Kd) of 7 nM for [3H]corticosterone and displayed a steroid hormone binding profile comparable to that of the native protein. Searches of the data banks revealed little overall similarity to other cloned genes. However, the amino acid sequence contains a dinucleotide-binding fingerprint. In conclusion, we have cloned the gene encoding the CBP from C. albicans and have shown that the expressed protein has the properties of the native CBP. A comparison of the cloned gene to members of the steroid-thyroid-retinoic acid receptor gene superfamily showed that CBP is unrelated to these hormone receptors.

Adrenal Cortex Hormones↗
PubMed1993

Regulation of vitamin D receptor abundance and responsiveness during differentiation of HT-29 human colon cancer cells.

We have studied the effects of 1,25-dihydroxyvitamin D [1,25-(OH)2D3] on cellular differentiation in the HT-29 human colon cancer cell line. Our aim was to evaluate the regulation of 1,25-dihydroxyvitamin D receptor (VDR) abundance and hormone responsiveness during the transition of rapidly proliferating to differentiated cells. Differentiation was induced by three means: cells were cultured in galactose-supplemented medium without glucose (GAL), grown on Matrigel-coated surfaces (MTG), or treated with 1,25(OH)2D3. Cell proliferation, assessed by [3H]thymidine incorporation, was equivalently inhibited by treatment with 1,25(OH)2D3, GAL or MTG. Differentiation was assessed by the induction of amino-oligo peptidase activity which was low in the proliferating cells. Following treatment with 1,25(OH)2D3, or growth in GAL or on MTG, amino-oligo peptidase activity increased 8- to 9-fold. The abundance of VDR measured by [3H]1,25(OH)2D3 binding, decreased to half without significant change in affinity, in cells differentiated by all three means compared to proliferating cells. Northern blot analyses of differentiated cells showed decreased steady-state levels of VDR messenger RNA (mRNA), indicating that all three treatments similarly decreased the abundance of VDR, at least in part, at the mRNA level. When exposed to 1,25(OH)2D3, the proliferating cells exhibited homologous up-regulation of VDR as well as the induction of 24-hydroxylase mRNA; the differentiated cells failed to exhibit both of these biological responses. Our findings demonstrate that 1,25(OH)2D3, GAL and MTG treatment all inhibit HT-29 cell proliferation and stimulate differentiation. Postproliferative differentiation achieved by the three approaches was associated with decreased VDR abundance, loss of VDR homologous up-regulation, and development of hormone unresponsiveness to 1,25(OH)2D3.

Calcitriol↗
PubMed1993

Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines.

It has been suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. In this study three human prostate carcinoma cell lines, LNCaP, DU-145, and PC-3, were examined both for the presence of specific 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] receptors (VDRs) and also employed to study the effects of hormone on cell proliferation and differentiation. Ligand binding experiments demonstrated classical VDR in all three cell lines examined with an apparent dissociation constant of 7.5, 5.4, and 6.3 x 10(-11) M for LNCaP, DU-145, and PC-3 cells, respectively. Corresponding binding capacity for the three prostate carcinoma cell lines were 27, 31, and 78 fmol/mg protein, respectively. The presence of VDR in the three cell lines was also confirmed by immunocytochemistry. In addition, one major 4.6-kilobase messenger RNA transcript hybridizing with a specific human VDR complementary DNA probe was identified in all three cell lines. Interestingly, both DU-145 and PC-3 but not LNCaP cell lines exhibited 1,25(OH)2D3-stimulated induction of 24-hydroxylase messenger RNA employed as a marker of 1,25(OH)2D3 action. Physiological levels of 1,25(OH)2D3 dramatically inhibited proliferation of the LNCaP and PC-3 cell lines. However, in spite of the presence of high affinity VDR, proliferation of DU-145 cells was not inhibited by 1,25(OH)2D3 at the doses tested. Treatment with 1,25(OH)2D3 caused a dose-dependent stimulation of prostate-specific antigen secretion by LNCaP cells. In conclusion, these results demonstrate that these three human prostate carcinoma cell lines all possess specific VDR and that 1,25(OH)2D3 treatment can elicit both an antiproliferative and a differentiating action on these cancer cells. The findings lend support to the hypothesis that vitamin D might exert beneficial actions on prostate cancer risk.

Blood↗
PubMed1993

Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving.

In studies to determine whether Saccharomyces cerevisiae produced estrogens, the organism was grown in culture media prepared using distilled water autoclaved in polycarbonate flasks. The yeast-conditioned media showed the presence of a substance that competed with [3H]estradiol for binding to estrogen receptors (ER) from rat uterus. However, it soon became clear that the estrogenic substance in the conditioned media was not a product of the yeast grown in culture, but was leached out of the polycarbonate flasks during the autoclaving procedure. [3H]Estradiol displacement activity was monitored by ER RRA, and the active substance was purified from autoclaved medium using a series of HPLC steps. The final purified product was identified as bisphenol-A (BPA) by nuclear magnetic resonance spectroscopy and mass spectrometry. BPA could also be identified in distilled water autoclaved in polycarbonate flasks without the requirement of either the organism or the constituents of the culture medium. Authentic BPA was active in competitive RRAs, demonstrating an affinity approximately 1:2000 that of estradiol for ER. In functional assays, BPA (10-25 nM) induced progesterone receptors in cultured human mammary cancer cells (MCF-7) at a potency of approximately 1:5000 compared to that of estradiol. The BPA effect on PR induction was blocked by tamoxifen. In addition, BPA (25 nM) increased the rate of proliferation of MCF-7 cells assessed by [3H]thymidine incorporation. Thus, BPA exhibited estrogenic activity by both RRA and two functional bioresponse assays. Finally, MCF-7 cells grown in media prepared with water autoclaved in polycarbonate exhibited higher progesterone receptor levels than cells.grown in media prepared with water autoclaved in glass, suggesting an estrogenic effect of the water autoclaved in polycarbonate. Our findings raise the possibility that unsuspected estrogenic activity in the form of BPA may have an impact on experiments employing media autoclaved in polycarbonate flasks. It remains to be determined whether BPA derived from consumer products manufactured from polycarbonate could significantly contribute to the pool of estrogenic substances in the environment.

Animals↗
PubMed1992

Cloning and characterization of the gene encoding the ADP-ribosylation factor in Candida albicans.

We have cloned and sequenced the gene (ARF) encoding the ADP-ribosylation factor (ARF) of Candida albicans. The gene contains an open reading frame of 537 nucleotides (nt) that codes for a protein with an Mr of 20,259. The C. albicans ARF gene is 67-70% identical at the nt level to other ARF sequences including those of humans; the deduced amino acid sequence of C. albicans ARF shows a 78-83% identity and 89-92% similarity to the other ARFs. Southern analysis of C. albicans genomic DNA suggested the presence of a second ARF gene. The presence of multiple ARF genes is a consistent finding among the other organisms previously shown to have ARFs.

ADP-Ribosylation Factors↗
PubMed1992

Mechanical properties of fibrin adhesives for blood vessel anastomosis.

Various methods have been used for anastomosing, or attaching, two ends of a severed blood vessel together. The most common method, suturing, is tedious, can be time-consuming, and requires special training in microvascular surgery. Other methods, such as mechanical devices and lasers, have some problems as well. The use of fibrin adhesives for blood vessel anastomosis might eliminate some of the current problems by allowing a quicker, simpler, and more reliable method of attachment. Although mechanical studies have been conducted to determine fibrin glue properties in shear, tensile, and burst tests; most of these studies have used skin or intestinal tissue. Therefore, to evaluate the feasibility of using fibrin glue as an adhesive for blood vessel anastomosis, the mechanical properties of blood vessels joined with fibrin glue were examined using tensile and burst tests. High and low fibrinogen concentrations were tested after 5- or 45-min time periods. In addition, three clinical methods of attachment were compared: end-to-end anastomosis, vessel overlapping, and suturing. In this study, because the adhesive strength was not found to increase significantly after 5 min, setting times for fibrin glue may be short enough to make it a clinical option when compared to suturing. In addition, the higher fibrinogen concentration did not result in a significantly higher adhesive strength, indicating that the lower concentration fibrin adhesives may be of comparable strength to the higher concentrations for clinical applications.

Anastomosis, Surgical↗
PubMed1992

Painless abruptio placentae associated with disseminated intravascular coagulation and syncope.

Abruptio placentae is a complication of pregnancy associated with significant morbidity and mortality for both fetus and mother. The presence of uterine pain and tenderness traditionally has been considered a useful criterion for distinguishing abruptio placentae from other causes of bleeding in pregnancy. We report the case of a 25-year-old woman who presented with painless abruptio placentae and coagulopathy during the second trimester. This case illustrates the importance of considering this diagnosis in pregnant patients with vaginal bleeding.

Abruptio Placentae↗
PubMed1992

Cyclic adenosine 3',5'-monophosphate up-regulates 1,25-dihydroxyvitamin D3 receptor gene expression and enhances hormone action.

We have previously shown that the abundance of vitamin D receptors (VDR) in cultured cells is increased by mitogens such as serum and growth factors, whereas activation of protein kinase-C (PK-C) causes inhibition of VDR gene expression. This study examines the effect of the cAMP-activated protein kinase-A (PK-A) second messenger system on VDR abundance and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] action. Elevation of intracellular cAMP levels in NIH-3T3 mouse fibroblasts by forskolin or (Bu)2cAMP caused a substantial (8- to 12-fold) increase in VDR abundance, as measured by ligand binding and Western blot analysis. The time course of the forskolin effect on VDR expression was complex. An early rise in VDR abundance occurred at 4 h, followed by a decrease and then a broad secondary rise at 18 h. At the mRNA level, forskolin caused a rapid rise in VDR transcripts after 1 h of exposure, a peak at 2 h, followed by a decline and a subsequent increase at 15 h. Activation of PK-C with the phorbol ester phorbol myristate acetate abolished the forskolin-induced increase in VDR protein and mRNA abundance. NIH-3T3 cells were stably transfected with phOC-CAT, a plasmid carrying a human osteocalcin promoter fragment containing the vitamin D response element fused to the reporter gene chloramphenicol acetyl transferase (CAT). 1,25-(OH)2D3 treatment of transfected cells induced a dose-dependent increase in CAT activity. Up- or down-regulation of VDR in these transfected cells by forskolin or phorbol myristate acetate pretreatment, respectively, resulted in corresponding enhancement or attenuation of 1,25-(OH)2D3-inducible CAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1992

Ultrastructure of peripheral neuropathy induced in rabbits by 2',3'-dideoxycytidine.

The nucleoside analogue, 2',3'-dideoxycytidine (ddC), an inhibitor of human immunodeficiency virus reverse transcriptase, mediates virologic and immunologic improvements in acquired immunodeficiency syndrome patients. However, in clinical studies ddC treatment is associated with a dose-limiting peripheral neuropathy. The purpose of this study was to characterize the ultrastructural features of the peripheral neuropathy induced in rabbits. Rabbits received 0, 10, 50, or 100 mg/kg/day of ddC for 18 weeks. A prominent ultrastructural change induced by ddC in sciatic nerve and ventral root was separation of myelin lamellae at the intraperiod line and vacuolation and fragmentation of myelin sheaths. Many demyelinated and remyelinated axons were observed. Although pathologic alterations in Schwann cells were evident by the presence of lipid droplets and myelin figures, their formation was not considered a primary event. Axons containing abnormal cytoplasmic components were occasionally observed. Other changes included redundance of Schwann cell basal lamina and the presence of lipid droplets and/or myelin figures within endoneurial fibroblasts and macrophages. The results of this study indicate that ddC induces a myelinopathy in rabbits characterized by myelin splitting and intramyelinic edema and an axonopathy that may be secondary to the myelin changes.

Animals↗
PubMed1991

Antiproliferative effect of interleukin-1 on human ovarian carcinoma cell line (NIH:OVCAR-3).

The human ovarian carcinoma cell line, NIH:OVCAR-3, possesses high affinity receptors for interleukin-1 (IL-1). Binding experiments with 125I-IL-1 alpha indicate a dissociation constant of approximately 55 pM and the presence of approximately 7800 receptors/cell. These receptors bind both IL-1 alpha and IL-1 beta and internalize IL-1. Proliferation is NIH:OVCAR-3 cells is inhibited by IL-1. Half-maximal inhibition is observed with 2-3 units/ml of IL-1 alpha or IL-1 beta. A maximal effect (80% inhibition of cell proliferation) is achieved by treatment of cells with greater than or equal to 10 units/ml of IL-1 for 3 days. The antiproliferative effect of IL-1 is blocked by IL-1 receptor antagonist. Light and electron microscopy studies show that IL-1 treatment causes cytopathological changes and a reduction in the number of mitotic figures in NIH:OVCAR-3. IL-1 stimulates prostaglandin E2 release by NIH:OVCAR-3 cells, but this response is unrelated to the antiproliferative effect of IL-1. Interferon-alpha A (IFN-alpha A) also inhibits growth of NIH:OVCAR-3 cells in a concentration-dependent manner. Combination of IFN-alpha A and IL-1 gives synergistic inhibition of NIH:OVCAR-3 cell proliferation. IL-1 alone or in combination with IFN-alpha A or other agents may be useful for treatment of human ovarian cancer.

Carcinoma↗
PubMed1991

The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hip.

Clinical criteria for the classification of patients with hip pain associated with osteoarthritis (OA) were developed through a multicenter study. Data from 201 patients who had experienced hip pain for most days of the prior month were analyzed. The comparison group of patients had other causes of hip pain, such as rheumatoid arthritis or spondylarthropathy. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop different sets of criteria to serve different investigative purposes. Multivariate methods included the traditional "number of criteria present" format and "classification tree" techniques. Clinical criteria: A classification tree was developed, without radiographs, for clinical and laboratory criteria or for clinical criteria alone. A patient was classified as having hip OA if pain was present in combination with either 1) hip internal rotation greater than or equal to 15 degrees, pain present on internal rotation of the hip, morning stiffness of the hip for less than or equal to 60 minutes, and age greater than 50 years, or 2) hip internal rotation less than 15 degrees and an erythrocyte sedimentation rate (ESR) less than or equal to 45 mm/hour; if no ESR was obtained, hip flexion less than or equal to 115 degrees was substituted (sensitivity 86%; specificity 75%). Clinical plus radiographic criteria: The traditional format combined pain with at least 2 of the following 3 criteria: osteophytes (femoral or acetabular), joint space narrowing (superior, axial, and/or medial), and ESR less than 20 mm/hour (sensitivity 89%; specificity 91%). The radiographic presence of osteophytes best separated OA patients and controls by the classification tree method (sensitivity 89%; specificity 91%). The "number of criteria present" format yielded criteria and levels of sensitivity and specificity similar to those of the classification tree for the combined clinical and radiographic criteria set. For the clinical criteria set, the classification tree provided much greater specificity. The value of the radiographic presence of an osteophyte in separating patients with OA of the hip from those with hip pain of other causes is emphasized.

Hip Joint↗
PubMed1991

Stimulation of 1,25-dihydroxyvitamin D3 receptor gene expression in cultured cells by serum and growth factors.

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in bone cells has been shown to vary in direct relation to the rate of cell proliferation. In this study we further explored this upregulation of VDR as it relates to the mitogenic response using NIH-3T3 mouse fibroblasts and MCF-7 human breast cancer cells as model systems. Serum and growth factors, such as EGF, high concentrations of insulin (2 microM), and IGF-I, were mitogenic and stimulated the proliferation of both cells types. These factors also caused significant increases in VDR levels as measured by ligand binding assays, which preceded the rise in cell proliferation rate measured by [3H]thymidine incorporation. Serum and growth factors increased the abundance of VDR but did not affect the concentrations of other steroid receptors in MCF-7 cells. Mouse cells have been reported to have several VDR mRNA transcripts. Our northern blot analysis revealed three mRNA species at approximately 7.5, 4.4, and 3 kb of which the 4.4 kb species was the most prominent and the 7.5 kb the least. Serum and growth factor stimulation of quiescent 3T3 cells led to significant increases in all the transcripts, suggesting that the upregulation occurs at the level of VDR mRNA expression. A time course analysis of serum stimulation in 3T3 cells showed that the mRNA species reached peak levels 4 h after serum addition. When serum stimulation was carried out in the presence of the protein synthesis inhibitor cycloheximide, the 3 kb transcript as well as the 7.5 kb transcript were superinduced but the stimulation of the 4.4 kb transcript was inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Fatal hyperammonemia resulting from a C-to-T mutation at a MspI site of the ornithine transcarbamylase gene.

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle in humans and is responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes. The cosegregation of the trait with a 5.8-kb abnormal MspI fragment in an affected family led us to hypothesize that this unexpected migration pattern was related to the mutation event in this particular family. Using polymerase chain reaction amplification of the specific mRNA derived from a post-mortem biopsy of the liver, we found that the MspI site located in the seventh exon of the gene was abolished and we finally identified a C-to-T transition at codon 225 of the cDNA, changing a proline to a leucine in the protein. Subsequent digestion of amplified exon 7 using the restriction enzyme MspI allowed direct screening for the mutant genotype during the next pregnancy. The present study supports the view that direct detection of the mutant genotype using either Southern blotting or digestion of amplified exons of the gene can contribute to genetic counselling in noninformative families. Finally, since MspI digestions are routinely performed for restriction fragment length polymorphism-based family studies in OTC deficiency, we suggest that the possible presence of the 5.8-kb abnormal fragment should be investigated on Southern blots of affected individuals.

Amino Acid Metabolism, Inborn Errors↗