Structure of the human biotinidase gene.

Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin. We have determined the structure of the human biotinidase gene. A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization. To obtain a clone containing the most 5' exon of the biotinidase cDNA, a human PAC library by PCR was screened. The human biotinidase gene is organized into four exons and spans at least 23 kb. The 5'-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element. The region from nt -600 to +400 has features of a CpG island and resembles a housekeeping gene promoter. The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency.

Mutations in the human biotinidase gene that cause profound biotinidase deficiency in symptomatic children: molecular, biochemical, and clinical analysis.

Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin biotin. The disorder can cause neurologic and cutaneous abnormalities that can be treated effectively with pharmacologic doses of biotin. We identified 21 mutations that cause profound biotinidase deficiency in 37 symptomatic children (30 different probands and 7 siblings), as well as provide relevant biochemical and clinical information for each child. The two most common mutations (G98:d7i3 and R538C) were found in 31 of 60 alleles (52%), whereas the remainder of the alleles are accounted for by the 19 other unique mutations. Serum samples were available from 18 children, of these 11 had no detectable cross-reacting material (CRM) to antibody prepared against normal human serum biotinidase, three had reduced quantities of CRM and four had normal quantities of CRM in serum. All of these mutations result in complete absence of biotinyl-transferase activity in serum. Two polymorphisms were also identified in normal individuals. It is apparent that a child who inherits any of these mutations, either in the homozygous state or in combination, can develop the clinical features of the disorder if untreated. There are, however, no clear genotype/phenotype correlations that would allow for the prediction of the type, severity, or age of onset of symptoms.

Mutation (Q456H) is the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States.

Biotinidase deficiency is an autosomal recessive disorder that can result in neurologic and cutaneous symptoms if not treated with biotin supplementation. We have identified the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. 1368A-->C results in a substitution of histidine for glutamine 456 (Q456H) in exon D of the biotinidase gene. This mutation was found in at least one allele in 14 unrelated children from 27 different families or 15 of 54 alleles studied (28%). This mutation was not identified in 41 normal adults using SSCA, nor was it found in 296 normal newborns using allele-specific oligonucleotide analysis, suggesting that this change is not a polymorphism. In addition, biochemical data from a child homozygous for Q456H suggest that the aberrant enzyme has very low biotinyl-hydrolase activity, lacks biotinyl-transferase activity, and is not recognized by antibody prepared to purified, normal human biotinidase. The ethnic backgrounds of the parents contributing the Q456H allele are varied but are generally northern European.

Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene.

Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100-->A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5'-3' scanning model, but is consistent with the exon definition model of RNA splicing.

Arg538 to Cys mutation in a CpG dinucleotide of the human biotinidase gene is the second most common cause of profound biotinidase deficiency in symptomatic children.

Biotinidase deficiency is an autosomal recessively inherited disorder in the recycling of the vitamin biotin. The most common mutation that causes profound biotinidase deficiency in symptomatic individuals is a deletion/insertion (G98:d7i3) that occurs in exon B of the biotinidase gene. We now report the second most common mutation, a C-to-T substitution (position 1612) in a CpG dinucleotide in exon D of the biotinidase gene. This mutation results in the substitution of a cysteine for arginine538 (designated R538C) and was found in 10 of 30 symptomatic children with profound biotinidase deficiency, 5 of whom also have the G98:d7i3 mutation. This mutation was not found in DNA samples from 32 individuals with normal biotinidase activity, but was found in one individual with enzyme activity in the heterozygous range. This mutation was not detected in 371 randomly selected, normal individuals using allele-specific oligonucleotide hybridization analysis. Aberrant biotinidase protein was not detectable in extracts of fibroblasts from a child who is homozygous for the R538C mutation, but was present in less than normal concentration in identical extracts treated with beta-mercaptoethanol. Because there is no detectable biotinidase protein in sera of children who are homozygous for the R538C mutation and in combination with the deletion/insertion mutation, the R538C mutation likely results in inappropriate intra- or intermolecular disulfide bond formation, more rapid degradation of the aberrant enzyme, and failure to secrete the residual aberrant enzyme from the cells into blood.

Deletion/insertion mutation that causes biotinidase deficiency may result from the formation of a quasipalindromic structure.

Biotinidase is responsible for recycling the vitamin biotin from biocytin that is formed after the proteolytic degradation of the biotin-dependent carboxylases. We have identified a deletion/insertion mutation within exon D of the human biotinidase gene in a child with biotinidase deficiency. The mutation causes a frame shift and premature termination which are predicted to result in a truncated protein. We propose that the mutation occurred during DNA replication by either of two mechanisms. Both mechanisms involve formation of a quasipalindromic hairpin loop in the template and dissociation of DNA polymerase alpha. This mutation supports the formation of palindromic structures as a possible cause of deletions in eukaryotes, and supports the proposal, derived from in vitro studies, that polymerase alpha may preferentially arrest or dissociate at specific template sequences.

Mutational hotspot in the human biotinidase gene causes profound biotinidase deficiency.

Biotinidase deficiency is an autosomal recessive inherited disorder that is characterized by neurological and cutaneous symptoms. Biotinidase-deficient children cannot recycle endogenous biotin, an essential water-soluble B vitamin. Biotin is covalently attached to epsilon-amino groups of lysyl residues of four carboxylases. These carboxylases are subsequently degraded to biocytin (biotin-epsilon-lysine). Biotinidase cleaves biocytin to biotin and lysine, thereby completing the biotin cycle. The symptoms of biotinidase deficiency can be resolved or prevented by treatment with biotin. Therefore, it is important that biotinidase deficiency is diagnosed early so that permanent neurological damage can be prevented. Many states and countries currently perform newborn screening for biotinidase deficiency. We have recently isolated and characterized the cDNA for normal human biotinidase and localized the gene to chromosome 3p25 (ref. 9). We have now identified the first mutation that causes profound biotinidase deficiency. It occurs in a distinct region of the gene that encodes the putative signal peptide. Fifty percent of symptomatic children studied have a 7-bp deletion coupled with a 3-bp insertion in at least one of their alleles of the biotinidase gene. This mutation appears to be a common cause of biotinidase deficiency in symptomatic children.

Reversal of ventricular contracture during hypothermic cardioplegic arrest.

Ventricular contracture was produced in isolated perfused rat hearts by a novel method using repeated administration of an anoxic cold hyperkalemic cardioplegia solution. Contracture could be reversed by reperfusion with the same solution, without calcium (Group 1), oxygenated (Group 2), or oxygenated and calcium free (Group 3). Group 1 hearts underwent partial reversal of contracture; in Group 2, contracture was reversed more completely, but the effect was transient. Hearts in Group 3 had contracture reversed completely to a level lower than prearrest end diastolic pressure. Hearts in contracture were profoundly depleted of high-energy phosphates (ATP, 9% of control; creatine phosphate, 27%) and the success of contracture reversal was paralleled by the extent to which each solution repleted ATP and PCr. Ventricular contracture produced by energy depletion is rapidly reversed by restoration of oxygen to the myocardium. Reducing extracellular calcium by acalcemic perfusion is ineffective as an isolated measure but is synergistic with reoxygenation and enhances contracture reversal.

Human serum biotinidase. cDNA cloning, sequence, and characterization.

Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnormalities, and can be successfully treated with biotin supplementation. Sequences of tryptic peptides of the purified human serum enzyme were used to design oligonucleotide primers for polymerase chain reaction amplification from human hepatic total RNA to generate putative biotinidase cDNA fragments. Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe revealed an open reading frame of 1629 bases encoding a protein of 543 amino acid residues, including 41 amino acids of a potential signal peptide. Comparison of the open reading frame with the known biotinidase tryptic peptides and recognition of the expressed protein encoded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA. Southern analyses suggested that biotinidase is a single copy gene and revealed that human cDNA probes hybridized to genomic DNA from mammals, but not from chicken or yeast. Northern analysis indicated the presence of biotinidase mRNA in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, and pancreas.

Optimization of coupled PCR amplification and cycle sequencing of cloned and genomic DNA.

We describe optimization of a coupled amplification and cycle sequencing (CAS) method for rapid characterization of cloned or genomic DNA. Our modification of this method, termed coupled PCR amplification and cycle sequencing (CPACS), utilizes commercially available reagents, does not require template purification and produces high-quality sequence ladders from nanogram quantities of complex genomic DNA. The reactions have been streamlined to permit automation. Finally, we show that the technique can be applied more efficiently in conjunction with the AutoTrans 350 Direct Transfer Electrophoresis System and 33P-labeled sequencing primers.

Relation of myocardial protection to cardioplegic solution pH: modulation by calcium and magnesium.

The relationship between myocardial preservation and cardioplegic solution pH was assessed in isolated, perfused rat hearts. A base solution without calcium or magnesium and the same solution containing 0.2 mmol/L ionized calcium or 16 mmol/L magnesium or both ions were studied at several values of pH between 6.8 and 8.7. Hearts were arrested at 8 degrees C by multidose infusions of these bicarbonate-buffered solutions bubbled with oxygen and a varying percentage of carbon dioxide to control pH. Diastolic tone (left ventricular balloon) and adenosine triphosphate (ATP) depletion during arrest both increased as the cardioplegic solution became more alkaline. Calcium increased these effects of pH. Magnesium weakened the effect of pH on diastolic tone, maintained ATP at all pH levels, and inhibited the effects of calcium on the relationships of pH to diastolic tone and ATP. When data from all solutions were considered together, ATP depletion was shown to be linearly related to diastolic tone. Calcium depressed functional recovery (left ventricular developed pressure during reperfusion expressed as a percentage of its prearrest value) at all pH levels. With the other solutions, recovery was similar and best within a broad and relatively alkaline pH range. With the solution containing calcium and magnesium, at pH levels of 8.28 +/- 0.02, 7.87 +/- 0.03, 7.58 +/- 0.02, 7.41 +/- 0.01, 7.06 +/- 0.02, and 6.80 +/- 0.01, recovery at 5 minutes of reperfusion was 101.4% +/- 3.7%, 102.9% +/- 2.8%, 107.3% +/- 3.7%, 102.8% +/- 2.9%, 91.8% +/- 3.6%, and 94.3% +/- 3.5%, respectively. This effect of alkalinity was short-lived. Extreme alkalinity of the base, acalcemic solution produced the calcium paradox, as reported previously. Good preservation of ATP by the most acid solutions did not predict good functional recovery. Magnesium increased the persistence of frequent extrasystoles during early reperfusion, but the effect was attenuated by calcium. The data support the inclusion of magnesium in cardioplegic solutions, particularly when they contain calcium, show that cardioplegic solution pH can have major effects on the arrested heart, and suggest that a relatively alkaline pH may modestly benefit functional recovery.

Erythropsial phototoxicity associated with nonultraviolet-filtering intraocular lenses.

Four patients with monoocular erythropsia (seeing red) caused by excessive exposure to bright light are presented. Each patient had a nonultraviolet (UV)-filtering intraocular lens (IOL) in the involved eye; the fellow eye was either phakic or had a UV-filtering IOL. Each patient had seen a definite red hue to lights (moon, automobile headlights, etc.) only at night after prior prolonged exposure to high-intensity sunlight and only in the eye with a non-UV-filtering IOL.

Myocardial preservation related to magnesium content of hyperkalemic cardioplegic solutions at 8 degrees C.

This study investigates whether the addition of magnesium to a hyperkalemic cardioplegic solution containing 0.1 mM ionized calcium improves myocardial preservation, and whether there is an optimal magnesium concentration in this solution. Isolated perfused rat hearts were arrested for two hours by this cardioplegic solution, which was fully oxygenated and infused at 8 degrees C every 15 minutes to simulate clinical conditions. The cardioplegic solution contained either 0, 2, 4, 8, 16, or 32 mM magnesium. At end-arrest, the myocardial creatine phosphate concentration (nanomoles per milligram of dry weight) was 20.7 +/- 2.1, 22.9 +/- 1.7, 24.8 +/- 2.0, 31.3 +/- 1.4, 33.1 +/- 1.8, and 31.6 +/- 0.8, respectively, in hearts given cardioplegic solution containing these magnesium concentrations. Thus, the concentration of creatine phosphate was significantly higher at end-arrest when the cardioplegic solution contained 8, 16, or 32 mM than 0 or 2 mM magnesium (p less than 0.002) or 4 mM magnesium (p less than 0.02), and highest with 16 mM magnesium. Also, creatine phosphate was more sensitive to the magnesium concentration of the cardioplegic solution than was end-arrest adenosine triphosphate levels, which did not differ among the experimental groups. Aortic flow, expressed as a percentage of prearrest aortic flow, was 60.3 +/- 5.0, 70.2 +/- 5.5, 71.6 +/- 4.4, 71.8 +/- 4.8, 81.0 +/- 5.0, and 71.8 +/- 5.3, respectively. The addition of magnesium to the cardioplegic solution improved recovery of aortic flow (p less than 0.05, 16 mM versus 0 mM magnesium). We conclude from these data that with deep myocardial hypothermia and at an ionized calcium concentration of 0.1 mM, the addition of magnesium, over a broad concentration range, improved preservation of myocardial creatine phosphate and, at a concentration of 16 mM, improved aortic flow. The optimal magnesium concentration in the cardioplegic solution was 16 mM.

Stride length and its determinants in humans, early hominids, primates, and mammals.

Primate stride lengths during quadrupedal locomotion are very long when compared to those of nonprimate quadrupedal mammals at the speed of trot/gallop transition. These exceptional lengths are a consequence of the relatively long limbs of primates and the large angular excursions of their limbs during quadrupedalism. When quadrupedal primates employ bipedal gaits they exhibit much lower angular excursions. Consequently their bipedal stride lengths do not appear to be exceptional in length when compared to other mammals. Angular excursions of the lower limbs of modern humans are not exceptionally large. However, when running, humans exhibit relatively long periods of flight (i.e., they have low duty factors) when compared to other mammals including primates. Because of these long periods of flight and their relative long lower limbs, humans have running stride lengths that are at the lower end of the range of stride lengths of quadrupedal primates. The stride length of the Laetoli hominid trails are evaluated in this context.

Mechanics of increased support of weight by the hindlimbs in primates.

Quadrupedal primates support most of their weight on their hindlimbs during locomotion. Neither the position of their center of gravity nor the average position of their foot contacts is substantially different from that of other quadrupeds supporting most of their weight on their forelimbs. Arguments are presented to support the theory that high levels of hindlimb retractor activity will produce this shift of support to the hindlimbs. If this muscular activity is appropriately timed, it will generate only low horizontal accelerations, which can be offset by small changes in the average position of the limbs. Estimates of muscular force are derived from force plate and kinematic data, which indicate that primates in fact do exhibit the postulated pattern of muscular activity. It is suggested that this shift occurs to reduce the compressive forces on the forelimbs.

Stresses on the limbs of quadrupedal primates.

Data is presented from eight primates on the ground reaction forces on the limbs during locomotion. These subjects supported from 30 to 45% of their body weight on their forelimbs. Other quadrupedal mammals support 55-60% of their body weight on their forelimbs. The increase of peak vertical force with speed varies greatly between the subjects. The variation in weight supported by the forelimbs and the peak forces on the forelimbs is proposed to correlate with variation in locomotor adaptations. It is suggested that the occurrence of bipedalism in primates represents the extreme expression of the tendency in primates to reduce the compressive forces on their forelimbs.