Roy A. Gravel

Neuronal apoptosis in sudden infant death syndrome.

Although evidence shows that victims of sudden infant death syndrome (SIDS) suffer repetitive episodes of hypoxemia, only subtle abnormalities have been found in their brains by light microscopy. The aim of the present study was to determine whether apoptosis, a form of cell death that can be triggered by hypoxemia and that leaves no scarring detectable by light microscopy, would be present in hypoxia-sensitive brain regions of SIDS victims. We looked for the presence of apoptosis with an in situ end-labeling method that detects DNA fragmentation. We studied 29 SIDS victims who were age-matched to nine control cases. We found significant neuronal apoptosis in 79% of the SIDS cases: 55% of the cases positive in the hippocampus and 96% positive in the brainstem. Whereas the distribution of apoptosis in the hippocampus was in hypoxia-sensitive subregions, the distribution in the brainstem was mostly in dorsal nuclei, including those involved with sensation in the face and position of the head (nucleus of the spinal trigeminal tract and vestibular nuclei). The control cases showed no significant apoptosis in the hippocampus and a mild degree in the brainstem in three cases. Our results indicate the occurrence of an acute insult at least several hours before death, an insult from which the infants had apparently recuperated. This suggests that SIDS victims suffered repeated apoptosis resulting in significant neuronal damage and, thus, functional loss in key brain regions. The involvement of specific nuclei in the brainstem may be linked to the fact that prone sleeping is a significant risk factor for SIDS. Enhanced neuronal death by apoptosis may thus have major implications for understanding the sequence of events leading to SIDS.

Lactic acidosis in biotin-responsive multiple carboxylase deficiency caused by holocarboxylase synthetase deficiency of early and late onset

Two patients with biotin-responsive multiple carboxylase deficiency, both presenting with predominant lactic acidosis, are reported. One with disease of early neonatal onset had considerable acute neurologic and persistent dermatologic abnormalities. The other, with late juvenile-onset disease, had chronic neurologic abnormalities without dermatologic findings. Early-onset cases generally have been associated with holocarboxylase synthetase deficiency, whereas those of juvenile onset have been characterized as representing defects in intestinal biotin absorption. However, enzyme analyses of fibroblasts from both patients, grown in biotin-deficient medium, revealed markedly diminished activities of pyruvate, propionyl-CoA, and beta-methylcrotonyl-CoA carboxylases, and all three enzymes showed normal activities after growth in biotin-rich medium. Furthermore, lymphoblast enzyme analysis in the patient with disease of early onset had previously revealed a defect in holocarboxylase synthetase, and fibroblast complementation studies showed that both patients belong to the bio complementation group. These findings indicate that considerable clinical heterogeneity exists among patients with holocarboxylase synthetase deficiency, an observation which does not permit differentiation of the biochemical forms of multiple carboxylase deficiency on the basis of age at onset and clinical presentation.

Cloning and sequence analysis of a cDNA encoding the β-subunit of mouse β-hexosaminidase

A cDNA encoding the prepro‐β‐polypeptide of mouse β‐hexosaminidase (Hex) was isolated from a mouse lymphoblast cDNA library. The cDNA contains an open reading frame corresponding to a polypeptide of 536 amino acids which shows 74% homology with the human prepro‐β‐polypeptide. An examination of the amino acid sequence identifies a putative signal peptide and five possible glycosylation sites, two of which are identical to the confirmed glycosylation sites of the human β‐chain. The amino acid sequence also shows a structurally similar though not identical site for internal cleavage responsible for the generation of mature βa‐ and βb‐polypeptides.

Prenatal Administration of Biotin in Biotin Responsive Multiple Carboxylase Deficiency

Summary: Biotin responsive multiple carboxylase deficiency was suspected in a third trimester conceptus on the basis of enzymatic confirmation in fibroblasts cultured from an earlier sibling who suffered a demise in the immediate neonatal period. Maternal urinary organic acid profile was normal throughout the final 4 wk of pregnancy. Oral administration of biotin, 10 mg/day to the mother resulted in a 100-fold increase in urinary biotin excretion within 7 days. Urinary biotin excretion over the subsequent 2 wk decreased steadily, suggesting either decreased maternal absorption or increased fetal sequestration.After the birth of nonidentical twins, cord blood and urinary organic acid profiles of the infants were normal. However, cord blood biotin concentration was 4 to 7-fold that of normal newborns. Subsequent enzymatic and genetic complementation studies utilizing cultured skin fibroblasts from the infants demonstrated one of them to be affected by the multiple carboxylase defect, although he was clinically and biochemically normal throughout the neonatal period.Thus, prenatal therapy of this inborn enzymatic defect can be safely and effectively accomplished by administration of pharmacologic biotin doses in the last month of pregnancy.Speculation: The affected infants excellent therapeutic response to pharmacologic doses of biotin, administered to the mother, and the absence of adverse effects in the mother and unaffected twin may eliminate the need for prenatal diagnosis in a pregnancy at risk for multiple carboxylase deficiency.

The amino-terminal sequences in the pro-α and -β polypeptides of human lysosomal β-hexosaminidase A and B are retained in the mature isozymes

The α‐ and β‐subunits of β‐hexosaminidase (β‐N‐acetylhexosaminidase, EC 3.2.1.52) are synthesized in the rough endoplasmic reticulum as prepropolypeptides. After the loss of the signal peptide and formation of enzymatically active dimers, the pro‐isoenzymes are transported through the Golgi and into the lysosome for proteolytic and glycolytic processing to their stable mature forms. Maturation includes the hydrolysis, and previously presumed loss, of small N‐terminal peptides from each propolypeptide. A recent report characterizing the processing of the β‐prepropolypeptide in β‐hexosaminidase from a human fibroblast cell line [(1989) J. Biol. Chem. 264, 3380–3384] reported that the small pro‐β peptide was retained through a disulfide bond in the mature subunit, and that it was glycosylated. We have confirmed this result in normal human tissue. However, we report a different N‐terminal for the mature pro‐β peptide. Furthermore, we have found that the pro‐α peptide is similarly retained in the mature α‐subunit through its single cysteine residue and that each pro‐peptide undergoes C‐terminal processing.

Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase

Abstract Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of β-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 %. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio -β-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.

Localization of the pro-sequence within the total deduced primary structure of human β-hexosaminidase B

The β subunit of β‐hexosaminidase (β‐N‐acetylhexosaminidase, EC 3.2.1.52) is synthesized in the rough endoplasmic reticulum as a prepropolypeptide. After the loss of the signal peptide and formation of an enzymatically active dimer, the pro‐enzyme is either secreted from the cell or transported into the lysosome for processing to its mature form. In order to characterize the early posttranslational events we have purified nearly 1 mg of pro‐hexosaminidase B from the NH4Cl containing medium of fibroblasts derived from a patient with the infantile form of Tay‐Sachs disease. The partial N‐terminal sequence was mapped to a position 42 residues C‐terminal to the first in‐frame ATG (Met residue) and 79 residues N‐terminal to the known mature N‐terminus. This position corresponds to that predicted for the cleavage of a 17 amino acid signal peptide generated through the use of the third rather than the first in‐frame ATG as the initiation site for protein synthesis.

Molecular Heterogeneity in O-Variant GM2 Gangliosidosis

The hydrolysis of GM2-ganglioside by β-hexosaminidase A (Hex A) is unique in its requirement for the proper synthesis and posttranslational processing of three different gene products. Two of these code for the pre-α and β polypeptides that combine to produce active pre-Hex A. The third is a specific Hex A activator protein required to complex with the natural substrate, GM2-ganglioside. Synthesis of mature Hex A and Hex B, the other major Hex isozyme in normal cells composed of only β-subunits, requires processing through the ER and Golgi apparatus with final sequestering in the lysosome. As a result of proteolytic processing in the lysosome the structures of the mature forms of Hex A and Hex B are α(βaβb) and 2(βaβb), where the pre-β chain is proteolytically split into covalently bound nonidentical βa and βb segments.

The Molecular Biology of β-Hexosaminidase: Localization of the Proteolytic Processing and Carbohydrate Containing Sites

The lysosomal storage diseases are inherited metabolic disorders characterized by the partial or total loss of the activity of specific lysosomal enzymes. This results in the irreversible accumulation of metabolites in lysosomes, ultimately leading to cell death. Many of these disorders are fatal, but less severe forms impair growth and development, often with neurological damage. The prototype lysosomal storage disease is Tay-Sachs disease, resulting from a defect involving β-hexosaminidase (N-acetylhexosaminidase, EC 3.2.1.52).

1192 PRENATAL THERAPY OF HOLOCARBOXYLASE SYNTHETASE DEFICIENCY

A 25y.o. G4P3Ab. woman who had previously delivered at least one and possibly two infants affected with a holocarboxylase synthetase deficiency was given biotin, 10mg p.o. daily over the last 4 wks. of pregnancy. No attempt at amniocentesis was made because of the late gestational stage of presentation. Twin male infants were delivered by elective C-section at 40 wks. gestation. Fibroblast cultures were begun immediately after birth, assay of which subsequently showed one infant to be normal and the other affected. However, daily monitoring of organic acids in urine and plasma over the first week of life showed no abnormality in either infant, both of whom appeared phenotypically normal. The subsequent clinical course of the affected baby has been reported elsewhere and documented clear biotin-responsiveness of the defect in vivo. Biotin measurements provided evidence that biotin administration during late pregnancy was effective in raising cord blood biotin to 4-7 fold control levels (30-48ng/ml vs. 7.34±0.55ng/ml). We conclude that it is possible to safely and effectively raise fetal blood biotin to therapeutic levels by oral administration of 10mg daily to the mother, in this case obviating the need for prenatal diagnosis and its attendant risks.