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1.  Four Children and Yale: The Making of a Human Geneticist 
Dr. Leon E. Rosenberg delivered the following presentation as the Grover Powers Lecturer on May 14, 2014, which served as the focal point of his return to his “adult home” as a Visiting Professor in the Department of Pediatrics. Grover F. Powers, MD, was one of the most influential figures in American Pediatrics and certainly the leader who created the modern Department of Pediatrics at Yale when he was recruited in 1921 from Johns Hopkins and then served as its second chairman from 1927 to 1951. Dr. Powers was an astute clinician and compassionate physician and fostered and shaped the careers of countless professors, chairs, and outstanding pediatricians throughout the country. This lectureship has continued yearly since it first honored Dr. Powers in 1956. The selection of Dr. Rosenberg for this honor recognizes his seminal role at Yale and throughout the world in the fostering and cultivating of the field of human genetics. Dr. Rosenberg served as the inaugural Chief of a joint Division of Medical Genetics in the Departments of Pediatrics and Internal Medicine; he became Chair when this attained Departmental status. Then he served as Dean of the Medical School from 1984 to 1991, before he became President of the Pharmaceutical Research Institute at Bristol-Myers Squibb and later Senior Molecular Biologist and Professor at Princeton University, until his recent retirement. Dr. Rosenberg has received numerous honors that include the Borden Award from the American Academy of Pediatrics, the McKusick Leadership Award from the American Society for Human Genetics, and election to the Institute of Medicine and the National Academy of Sciences.
PMCID: PMC4144292  PMID: 25191153
5.  Intestinal Transport of Cystine and Cysteine in Man: Evidence for Separate Mechanisms* 
Cystine and cysteine are transported by energy-dependent, mediated processes in human gut. When either of these amino acids is transported, only cysteine is recovered intracellularly, indicating that cystine is reduced to cysteine after achieving an intracellular location. In contrast to results with cystine, cysteine uptake is not defective in gut from cystinuric patients, nor do lysine and arginine compete with cysteine for transport. It is, therefore, concluded that cystine and cysteine are transported by different mechanisms, and that only the cystine transport mechanism is defective in cystinuria.
PMCID: PMC297017  PMID: 6018747
8.  Affinity of Cystathionine β-Synthase for Pyridoxal 5′-Phosphate in Cultured Cells 
Journal of Clinical Investigation  1980;66(2):188-193.
Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine β-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5′-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5′-phosphate fell by >95%, and saturation of cystathionine β-synthase apoenzyme with pyridoxal 5′-phosphate decreased from a predepletion value of 70% to <10%. When such depleted cells were grown in media containing pyridoxal concentrations ranging from 0 to 1,000 ng/ml, cellular pyridoxal 5′-phosphate reached a maximum of 30 ng/mg cell protein at a medium pyridoxal concentration of 100 ng/ml. Maximal saturation of aposynthase with coenzyme in control cells was reached at a medium pyridoxal concentration of 10 ng/ml. In contrast, maximal saturation of residual aposynthase in cells from an in vivo responsive patient was achieved at a medium pyridoxal concentration of 25-50 ng/ml, whereas that from cells from an in vivo unresponsive patient was reached at 100 ng/ml. Estimates of the affinity of control and mutant cystathionine β-synthase for pyridoxal 5′-phosphate in cell extracts supported the differences observed in intact cells. The apparent Km of cystathionine β-synthase for pyridoxal 5′-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the Km for pyridoxal 5′-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine β-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5′-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5′-phosphate.
PMCID: PMC371697  PMID: 7400312
9.  Inherited Methylmalonyl CoA Mutase Apoenzyme Deficiency in Human Fibroblasts 
Journal of Clinical Investigation  1980;65(3):690-698.
We have measured and characterized methylmalonyl coenzyme A (CoA) mutase activity in extracts of cultured human fibroblasts from 23 patients with inherited deficiency of the mutase apoenzyme and from eight obligate heterozygotes for this defect. The mutant cell lines fall into two categories. Those without detectable residual mutase activity in cell extracts (>0.1% of control), and whose ability to utilize propionate in intact cells is refractory to supplementation of the culture medium with hydroxocobalamin, are designated mut° mutants. Those with detectable residual activity in cell extracts (∼0.5-50% of control), and whose ability to utilize propionate in intact cells in markedly increased by hydroxocobalamin supplementation, are designated mut− mutants. The mutant enzyme in the mut− mutants exhibits a 50- to 5,000-fold elevated Michaelis constant (Km) for adenosylcobalamin in vitro, a normal Km for methylmalonyl CoA, and a strikingly reduced thermal stability at 45°C relative to control. Mutase from one mut− mutant turns over at a rate three to four times that of control enzyme when cells are grown in hydroxocobalamin-supplemented medium.
To detect heterozygous carriers of mutant mut alleles, we compared mutase activity in fibroblast extracts from four controls with that from eight parents of either mut° or mut− mutants. After cell growth in either unsupplemented or hydroxocobalamin-supplemented medium, activity in cell lines from heterozygotes was reduced to 47 or 37% of the mean control activities, respectively. We also examined the effect of adenosylcobalamin concentration on reaction kinetics of mutase from heterozygote cell lines. All four cell lines from parents of mut− mutants exhibited complex enzyme kinetics; ∼80% of mutase activity demonstrated a Km indistinguishable from control, whereas a smaller component of activity exhibited a Km similar to the abnormal Km expressed by the mut− propositus in each family. In two families with a mut° propositus, mutase from three of the four parents exhibited only the normal Km for adenosylcobalamin, whereas mutase from one parent displayed complex kinetics, indicating expression of both a normal allele (mut+) and a mutant allele with an abnormal Km. From these studies, we conclude that mut mutants reflect mutations at the autosomal gene locus for the methylmalonyl CoA mutase apoenzyme; that mut°, mut−, and mut+ alleles at this locus are codominantly expressed; and that some mut mutants may be genetic compounds, inheriting two different mut° or mut− alleles from their parents.
PMCID: PMC371411  PMID: 6101601
10.  Cobalamin Binding and Cobalamin-Dependent Enzyme Activity in Normal and Mutant Human Fibroblasts 
Journal of Clinical Investigation  1978;62(5):952-960.
We have studied the intracellular binding of radioactive cobalamin by normal cultured human fibroblasts grown in medium containing [57Co]-cobalamin. We have also assessed the significance of defects in this binding activity exhibited by two classes of human mutants (cbl C and cbl D) each characterized by pleiotropic deficiencies in the accumulation and retention of cobalamin, in the synthesis of cobalamin coenzymes, and accordingly, in the holoenzyme activities of both cobalamin-dependent enzymes, 5-methyltetrahydrofolate:homocysteine methyltransferase and methylmalonyl-CoA mutase. Based on the coincidence of [57Co]cobalamin binding and cobalamin-dependent enzyme activities after Sephadex G-150 chromatography and polyacrylamide gel electrophoresis, we conclude that, as in rat liver, the intracellular binding of labeled cobalamin by normal fibroblasts reflects the attachment of the vitamin to the cobalamin-dependent methyltransferase and mutase. Whereas cbl C cells are completely deficient in the binding of [57Co]cobalamin to either enzyme, fibroblasts which bear the phenotypically similar but genetically distinct cbl D mutation retain some binding activity, and accordingly, have higher holomethyltransferase and holomutase activities than do cbl C cells. The defect in [57Co]-cobalamin binding exhibited by both cbl C and cbl D fibroblasts is almost certainly not a result of mutations which affect the methyltransferase or mutase apoenzymes, since the electrophoretic mobilities and the affinities of these enzymes for their respective cobalamin coenzymes are indistinguishable from those in control cell extracts. These results suggest that both the cbl C and cbl D mutations affect some enzymatic step(s) which converts newly taken up cobalamin to a form capable of being bound by the two cobalamin-dependent enzymes.
PMCID: PMC371853  PMID: 30783
11.  Heterozygote Expression in Propionyl Coenzyme A Carboxylase Deficiency 
Journal of Clinical Investigation  1978;62(5):931-936.
We measured propionyl coenzyme A carboxylase (PCC) activity in extracts of skin fibroblasts and peripheral blood leukocytes from controls and obligate heterozygotes for PCC deficiency. 6 heterozygotes were from the pcc A complementation group; 12 were from the other major complementation group, designated pcc C. Mean PCC activity in fibroblast extracts from pcc A heterozygotes was 52% of that in controls, whereas mean PCC activity in pcc C heterozygotes was indistinguishable from that of controls. Similar results were obtained with extracts of peripheral blood leukocytes. In none of eight families (three pcc A and five pcc C) in which PCC activity was studied in both parents of an affected child were significant intrafamilial differences observed. The activities of two other mitochondrial enzymes (β-methyl-crotonyl CoA carboxylase and glutamate dehydrogenase) were comparable in controls and both groups of heterozygotes. Whereas the data from pcc A heterozygotes are consistent with expected gene dosage effects, those from pcc C heterozygotes are not. Inasmuch as mammalian PCC is a large molecular weight tetramer, each protomer of which is probably composed of two nonidentical subunits, the latter results are most consistent with unbalanced rates of synthesis and(or) degradation of the two subunits in normal cells with compensatory balancing in pcc C heterozygotes.
PMCID: PMC371850  PMID: 711858
12.  Homocystinuria 
Journal of Clinical Investigation  1978;61(3):645-653.
We have compared in vivo pyridoxine responsiveness with in vitro cystathionine β-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5′-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five- and sevenfold stimulation of cystathionine β-synthase activity with added pyridoxal-5′-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two- and fivefold with the added cofactor. Cystathionine β-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5′-phosphate were estimated and thermostability (54°C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine β-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5′ phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activity in vitro or with stimulation of in vitro enzyme activity by cofactor.
PMCID: PMC372577  PMID: 641146
13.  Binding and Uptake of Transcobalamin II by Human Fibroblasts 
Journal of Clinical Investigation  1978;61(1):133-141.
We have used purified, 125I-labeled human transcobalamin II (TC II), saturated with cobalamin (Cbl), to study the uptake process for the TC II-Cbl complex by intact normal cultured human skin fibroblasts. We have also investigated the possibility that a defect in one step of this process underlies that inborn error of Cbl metabolism—designated cbl C—in which mutant cells are unable to retain Cbl intracellularly or convert it to its coenzyme forms. TC II-Cbl binding at 4°C reached a plateau after 3-4 hr; 95% of the bound 125I was releasable with trypsin. Binding of TC II-Cbl at 4°C could be inhibited by human and rabbit TC II-Cbl and human TC II devoid of Cbl but not by other Cbl-binding proteins, albumin, or free Cbl. Specific binding reached saturation at ≅5 ng TC II/ml (0.13 nM) and could be inhibited by ethylene glycol-bis (β-aminoethyl ether) N,N,N′,N′- tetraacetic acid. At 37°C, the TC II-Cbl complex was internalized as shown by a progressive decrease in the trypsin-releasable fraction of bound 125I. After 2 h at 37°C, increasing amounts of acid-soluble 125I were found in the incubation medium indicating that the labeled TC II was being degraded. Chloroquine, an inhibitor of lysosomal proteolysis, prevented this degradation. The binding, internalization, and degradation of TC II-Cbl by cbl C cells was indistingusihable from that by control cells. Our studies provide additional support for the concepts: (a) that the TC II-Cbl complex binds to a specific cell surface receptor through a site on the TC II; (b) that the interaction between the receptor and TC II is calcium dependent; (c) that the TC II-Cbl is internalized via endocytosis; (d) that the degradation of TC II and release of Cbl from the complex occurs in lysosomes. We also conclude that the defect in cbl C must reside at some step beyond this receptor-mediated uptake process.
PMCID: PMC372521  PMID: 618908
14.  Transport of dibasic amino acids, cystine, and tryptophan by cultured human fibroblasts: absence of a defect in cystinuria and Hartnup disease 
Journal of Clinical Investigation  1972;51(8):2130-2142.
Transport of lysine, arginine, cystine, and tryptophan was studied in cultured skin fibroblasts from normal controls and from patients with cystinuria and Hartnup disease. Each of these amino acids was accumulated against concentration gradients by energy-dependent, saturable mechanisms. Lysine and arginine were each transported by two distinct processes which they shared with each other and with ornithine. In contrast, cystine was taken up by a different transport system with no demonstrable affinity for the dibasic amino acids. The time course and Michaelis-Menten kinetics of lysine and cystine uptake by cells from three cystinuric patients differed in no way from those found in control cells. Similarly, the characteristics of tryptophan uptake by cells from a child with Hartnup disease were identical to those noted in control cells. These findings indicate that the specific transport defects observed in gut and kidney in cystinuria and Hartnup disease are not expressed in cultured human fibroblasts, thus providing additional evidence of the important role that cellular differentiation plays in the regulation of expression of the human genome.
PMCID: PMC292370  PMID: 5054467
15.  Inherited propionyl-CoA carboxylase deficiency in “ketotic hyperglycinemia” 
Journal of Clinical Investigation  1971;50(1):127-130.
Cultured fibroblasts from a young girl with ketotic hyperglycinemia were unable to oxidize propionate-14C to 14CO2, but oxidized methylmalonate-14C and succinate-14C normally. This block in propionate catabolism was shown to result from a lack of propionyl-CoA carboxylase activity. The carboxylase deficiency was not due to the presence of an intracellular inhibitor and it was not corrected by biotin, a known cofactor for the enzyme. Both of her parents' fibroblasts had approximately 50% of normal propionyl-CoA carboxylase activity. These results demonstrate that ketotic hyperglycinemia and propionicacidemia are the same disease, caused by a mutation of the propionyl-CoA carboxylase apoenzyme, which is inherited as an autosomal recessive trait. This enzymatic localization provides an explanation for the remarkable clinical and chemical similarity between ketotic hyperglycinemia and methylmalonicaciduria and offers a potential means of antenatal detection of this disorder.
PMCID: PMC291900  PMID: 5101292
16.  Renal and intestinal hexose transport in familial glucose-galactose malabsorption 
Journal of Clinical Investigation  1970;49(3):576-585.
Glucose transport by jejunal mucosa in vitro and kidney in vivo was investigated in a 3 yr old patient with congenital glucose-galactose malabsorption, her family, and 16 normal volunteers. Glucose transport by normal human jejunal mucosa was concentrative, saturable, sodium and energy dependent, and exhibited competitive inhibition. Biopsy specimens from six normal controls and an asymptomatic 5 yr old brother of the proband accumulated glucose to concentrations 16 times that in the incubation medium. The proband's mucosa was unable to concentrate glucose throughout a 60 min incubation period. Both of her parents and a half sister demonstrated impaired glucose transport. Their values fell between normal and those of the proband. Influx of glucose was impaired but efflux of glucose from the mucosa of these three heterozygotes was identical with that in three normal controls. A kinetic analysis indicated a reduced capacity (Vmax), but a normal affinity (Km) for glucose transport by their intestinal mucosa. All subjects accumulated fructose similarly.
Renal glucose transport was investigated using renal glucose titration techniques. A partial defect in renal glucose reabsorption was found in the proband. Her brother's titration curve was similar to that of seven normal volunteers.
We conclude that familial glucose-galactose malabsorption is inherited as an autosomal recessive trait, that heterozygotes for this disorder are detectable and demonstrate a reduced capacity for glucose transport, and that absent intestinal glucose transport is accompanied by partial impairment of renal glucose transport.
PMCID: PMC322506  PMID: 5415683
17.  Familial renal glycosuria: a genetic reappraisal of hexose transport by kidney and intestine 
Journal of Clinical Investigation  1969;48(10):1845-1854.
Renal glucose titration studies were carried out in 10 members of two pedigrees with familial renal glycosuria to test the accepted hypothesis of autosomal dominant inheritance and to investigate the genetic significance of “type A” and “type B” renal glycosuria. In one family, a brother and sister each had a moderately reduced threshold and tubular maximum for glucose (type A), but both of their parents reabsorbed glucose normally. In the second family, two brothers had severe type A renal glycosuria, their mother and one brother had a mild type A defect, and another brother demonstrated a reduced threshold, an exaggerated splay, and a normal tubular maximum, indicative of type B glycosuria.
Hexose transport by intestinal mucosa was also investigated in controls and in the three brothers with the most severe renal glycosuria. D-glucose-14C and 3-O-methylglucose-14C were accumulated by jejunal mucosa from controls by processes which were saturable and concentrative. No differences in hexose transport were observed in the patients with renal glycosuria.
We conclude that familial renal glycosuria can be inherited as an autosomal recessive trait; that mild and severe type A renal glycosuria and type B renal glycosuria can occur in the same pedigree; and that defective reabsorption of glucose by the kidney need not be accompanied by abnormalities in intestinal glucose transport. These findings indicate that glucose transport in the gut and kidney are not mediated by identical mechanisms, and that several different mutations are responsible for the phenotypic variability in familial renal glycosuria.
PMCID: PMC322420  PMID: 5822589
18.  Dietary perturbation of calcium metabolism in normal man: compartmental analysis 
The effect of dietary calcium intake on calcium metabolism was studied in eight normal volunteers by multicompartmental analysis of radiocalcium and balance data. In paired studies of six normal subjects on normal and high or low calcium intakes, necessary and sufficient criteria were used to determine changes in calcium metabolic parameters produced by alterations in dietary calcium. These changes involved gastrointestinal calcium absorption rate, renal and endogenous fecal rate constants, and bone resorption rate. Bone accretion rate and compartment sizes need not change between the paired studies. The changes of parameters involving kidney, gut, and bone were in a direction to support calcium homeostasis and were compatible with the pattern of changes produced by parathyroid hormone. However, the source of the stimulus for hormone secretion was not apparent since plasma calcium concentrations showed no significant difference between paired studies. The implications of these findings relative to control of hormone secretion, calcium regulatory mechanisms, and metabolic bone disease are discussed.
PMCID: PMC322192  PMID: 5765028

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