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1.
J Med Genet ; 39(9): 623-33, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12205104

ABSTRACT

Fraser syndrome is characterised by cryptophthalmos, cutaneous syndactyly, malformations of the larynx and genitourinary tract, craniofacial dysmorphism, orofacial clefting, mental retardation, and musculoskeletal anomalies. The inheritance is autosomal recessive. No diagnostic cytogenetic abnormalities have been documented in affected patients, and no molecular genetic studies have been reported. We have reviewed 117 cases diagnosed as Fraser syndrome or cryptophthalmos published since the comprehensive review of Thomas et al in 1986 in order to validate the published diagnostic criteria and to delineate the phenotype associated with this syndrome. Our series showed more females (57/117) than males and consanguinity was present in 29/119 (24.8%). Eighty-eight patients satisfied the diagnostic criteria for Fraser syndrome (75%). Cryptophthalmos was present in 103/117 (88%), syndactyly in 72/117 (61.5%), and ambiguous genitalia in 20/117 (17.1%). Ear malformations were recorded in 69/117 (59%), and renal agenesis in 53/117 (45.3%). Use of the published diagnostic criteria excluded several patients with cryptophthalmos and one or more physical feature(s) consistent with Fraser syndrome. The frequency of additional anomalies in our series was also higher than previously reported (for example, imperforate anus or anal stenosis were found in 34/117 (29%) compared with 2/124 (2%) in the series of Thomas et al (1986) and choanal stenosis or atresia was present in 7/117 (6%) compared to 0/124. These findings emphasise the clinical variability associated with Fraser syndrome and support genetic heterogeneity of the syndrome. We also noted patterns of anomalies (for example, bicornuate uterus with imperforate anus or anal stenosis and renal malformations) that are found in other syndromes and associations without cryptophthalmos, suggesting that common modifier genes may explain some of the phenotypic variation in Fraser syndrome.


Subject(s)
Abnormalities, Multiple/pathology , Eye Abnormalities , Eyelids/abnormalities , Syndactyly/pathology , Abnormalities, Multiple/genetics , Diagnosis, Differential , Female , Humans , Male , Phenotype , Pregnancy , Pregnancy Complications , Sex Factors , Syndrome
2.
Hum Genet ; 109(3): 311-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11702212

ABSTRACT

We screened 120 children with sporadic multiple congenital anomalies and either growth or mental retardation for uniparental disomy (UPD) or subtelomeric deletions. The screening used short tandem repeat polymorphisms (STRP) from the subtelomeric regions of 41 chromosome arms. Uninformative marker results were reanalyzed by using the next available marker on that chromosome arm. In total, approximately 25,000 genotypes were generated and analyzed for this study. Subtelomeric deletions of 1 Mb in size were excluded for 27 of 40 chromosome arms. Among the 120 subjects none was found to have UPD, but five subjects (4%, 95% confidence interval 1-9%) were found to have a deletion or duplication of one or more chromosome arms. We conclude that UPD is not a frequent cause of undiagnosed multiple congenital anomaly syndrome. In addition, we determined that 9p and 7q harbor chromosome length variations in the normal population. We conclude that subtelomeric marker analysis is effective for the detection of subtelomeric duplications and deletions, although it is labor intensive. Given a detection rate that is similar to prior studies and the large workload imposed by STRPs, we conclude that STRPs are an effective, but impractical, approach to the determination of segmental aneusomy given current technology.


Subject(s)
Abnormalities, Multiple/genetics , Chromosome Aberrations , Chromosome Deletion , Telomere/genetics , Aneuploidy , Child , Female , Genetic Markers , Growth Disorders/genetics , Humans , Intellectual Disability/genetics , Male , Polymorphism, Genetic , Tandem Repeat Sequences
3.
Blood ; 97(1): 327-9, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11133779

ABSTRACT

Sandhoff disease is a lysosomal storage disorder characterized by G(M2) ganglioside accumulation in the central nervous system (CNS) and periphery. It results from mutations in the HEXB gene, causing a deficiency in beta-hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ]) independently have been shown to extend life expectancy in a mouse model of Sandhoff disease. The efficacy of combining these 2 therapies was evaluated. Sandhoff disease mice treated with BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into 2 groups on the basis of their donor bone marrow-derived CNS enzyme levels, the high enzyme group exhibited a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy may therefore be the strategy of choice for treating the infantile onset disease variants.


Subject(s)
1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/therapeutic use , Bone Marrow Transplantation , Sandhoff Disease/therapy , Animals , Brain/metabolism , Diagnostic Techniques, Neurological , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Glycosphingolipids/metabolism , Hexosaminidase B , Mice , Spinal Cord/metabolism , Survival Rate , beta-N-Acetylhexosaminidases/metabolism
4.
Am J Med Genet ; 95(3): 266-8, 2000 Nov 27.
Article in English | MEDLINE | ID: mdl-11102933

ABSTRACT

The clinical presentation of mitochondrial DNA (mtDNA) disorders is quite diverse. Very often, the initial symptoms do not fit a specific disease, and diagnosis is difficult to make. We describe a patient who presented with macrocytic anemia. Extensive biochemical and clinical work-up failed to provide an etiology for the macrocytic anemia. The patient over the course of 6 years developed gait problems, exercise intolerance, episodic vomiting, short stature, dermatological problems, and recurrent infection. At age 8 years she had encephalopathy with ataxia and dysphagia. The presence of elevated lactate, bilateral basal ganglia calcification, and ragged red fibers led to mtDNA mutational analysis. A novel 4.4-kb deletion from nucleotide position 10,560 to nucleotide position 14, 980 was identified in muscle biopsy. The same heteroplasmic mtDNA deletion was present in blood, buccal cells, and hair follicles, but not in mother's blood, consistent with sporadic mutation in the patient. This case emphasizes the importance of considering mtDNA disorder in patients with multisystemic symptoms that cannot be explained by a specific diagnosis.


Subject(s)
Anemia, Macrocytic/etiology , DNA, Mitochondrial/genetics , Anemia, Macrocytic/genetics , Anemia, Macrocytic/therapy , Blood Transfusion , Child , DNA Mutational Analysis , DNA, Mitochondrial/adverse effects , DNA, Mitochondrial/metabolism , Diagnosis, Differential , Female , Gene Deletion , Genetic Heterogeneity , Humans , Leukocytes , Muscles , Neutropenia/etiology , Neutropenia/genetics , Neutropenia/therapy , Syndrome , Tissue Distribution
5.
Proc Natl Acad Sci U S A ; 97(20): 10954-9, 2000 Sep 26.
Article in English | MEDLINE | ID: mdl-11005868

ABSTRACT

Sandhoff disease is a lysosomal storage disorder characterized by the absence of beta-hexosaminidase and storage of G(M2) ganglioside and related glycolipids in the central nervous system. The glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. In symptomatic Sandhoff disease mice, apoptotic neuronal cell death was prominent in the caudal regions of the brain. cDNA microarray analysis to monitor gene expression during neuronal cell death revealed an upregulation of genes related to an inflammatory process dominated by activated microglia. Activated microglial expansion, based on gene expression and histologic analysis, was found to precede massive neuronal death. Extensive microglia activation also was detected in a human case of Sandhoff disease. Bone marrow transplantation of Sandhoff disease mice suppressed both the explosive expansion of activated microglia and the neuronal cell death without detectable decreases in neuronal G(M2) ganglioside storage. These results suggest a mechanism of neurodegeneration that includes a vigorous inflammatory response as an important component. Thus, this lysosomal storage disease has parallels to other neurodegenerative disorders, such as Alzheimer's and prion diseases, where inflammatory processes are believed to participate directly in neuronal cell death.


Subject(s)
Bone Marrow Transplantation , Microglia/pathology , Neurons/pathology , Sandhoff Disease/pathology , Sandhoff Disease/therapy , Animals , Humans , Inflammation , Mice , Microglia/metabolism , Microglia/physiology , Neurons/metabolism , Neurons/physiology , Sandhoff Disease/metabolism , Sandhoff Disease/physiopathology , Transplantation, Homologous , beta-N-Acetylhexosaminidases/metabolism
6.
Am J Hum Genet ; 66(2): 419-27, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10677301

ABSTRACT

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include developmental and growth retardation. Current microscopic techniques are useful for the detection of such aberrations but have a limit of resolution that is above the threshold for phenotypic effect. We hypothesized that a genomewide microsatellite screen could detect chromosomal aberrations that were not detected by standard cytogenetic techniques in a portion of these individuals. To test this hypothesis, we performed a genomewide microsatellite screen of patients, by use of a currently available genetic-marker panel that was originally designed for meiotic mapping of Mendelian traits. We genotyped approximately 400 markers on 17 pairs of parents and their children who had normal karyotypes. By using this approach, we detected and confirmed two cases of segmental aneusomy among 11 children with multiple congenital anomalies. These data demonstrate that a genomewide microsatellite scan can be used to detect chromosomal aberrations that are not detected by microscopic techniques.


Subject(s)
Chromosome Aberrations/genetics , Genetic Testing/methods , Genome, Human , Microsatellite Repeats/genetics , Abnormalities, Multiple/genetics , Alleles , Child , Female , Gene Duplication , Genotype , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Meiosis/genetics , Nuclear Family , Pilot Projects , Reproducibility of Results , Sequence Deletion/genetics
7.
Acta Neuropathol ; 99(2): 161-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10672323

ABSTRACT

Tissue distribution of beta-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl beta-D-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (Hexa-/-, Hexb-/-, Gm2a-/- and Hexa-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of beta-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and Hexa-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or Hexa-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/- +WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.


Subject(s)
Bone Marrow Transplantation/pathology , Brain/pathology , G(M2) Ganglioside/analysis , Gangliosidoses, GM2/pathology , beta-N-Acetylhexosaminidases/metabolism , Animals , Brain/enzymology , Epithelial Cells/enzymology , Epithelial Cells/pathology , G(M2) Ganglioside/deficiency , G(M2) Ganglioside/genetics , Gangliosidoses, GM2/enzymology , Hexosaminidase A , Hexosaminidase B , Kidney Tubules/enzymology , Kidney Tubules/pathology , Liver/enzymology , Liver/pathology , Macrophages/enzymology , Macrophages/pathology , Mice , Mice, Knockout , Mice, Transgenic , Neurons/enzymology , Neurons/pathology , Spleen/enzymology , Spleen/pathology , beta-N-Acetylhexosaminidases/deficiency , beta-N-Acetylhexosaminidases/genetics
8.
Glycobiology ; 10(12): 1249-58, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11159916

ABSTRACT

Glycosphingolipids (GSLs) are plasma membrane components of every eukaryotic cell. They are composed of a hydrophobic ceramide moiety linked to a glycan chain of variable length and structure. Once thought to be relatively inert, GSLs have now been implicated in a variety of biological processes. Recent studies of animals rendered genetically deficient in various classes of GSLs have demonstrated that these molecules are important for embryonic differentiation and development as well as central nervous system function. A family of extremely severe diseases is caused by inherited defects in the lysosomal degradation pathway of GSLs. In many of these disorders GSLs accumulate in cells, particularly neurons, causing neurodegeneration and a shortened life span. No effective treatment exists for most of these diseases and little is understood about the mechanisms of pathogenesis. This review will discuss the development of a new approach to the treatment of GSL storage disorders that targets the major synthesis pathway of GSLs to stem their cellular accumulation.


Subject(s)
1-Deoxynojirimycin/analogs & derivatives , 1-Deoxynojirimycin/pharmacology , Glycosphingolipids/antagonists & inhibitors , Glycosphingolipids/biosynthesis , Lysosomal Storage Diseases/drug therapy , Propanolamines/pharmacology , Pyrrolidines/pharmacology , 1-Deoxynojirimycin/therapeutic use , Clinical Trials as Topic , Humans , Propanolamines/therapeutic use , Pyrrolidines/therapeutic use
9.
Am J Med Genet ; 86(4): 312-5, 1999 Oct 08.
Article in English | MEDLINE | ID: mdl-10494084

ABSTRACT

Cartilage hair hypoplasia (CHH), or metaphyseal dysplasia McKusick type, classically comprises short stature and scant fine hair. In this skeletal dysplasia there is a high incidence of immune deficiency and Hirschsprung disease, as well as a higher rate of malignancy. Clinical findings may be subtle in young children, and radiographic changes may be elusive. We present four children below age 2 in whom the clinical diagnosis of CHH was confirmed radiographically. We emphasize radiologically and clinically discernable anterior angulation of the entire sternum, a sign not previously described in this dysplasia.


Subject(s)
Cartilage/abnormalities , Hair/abnormalities , Osteochondrodysplasias/diagnostic imaging , Cartilage/diagnostic imaging , Consanguinity , Female , Genes, Recessive , Humans , Immunologic Deficiency Syndromes/genetics , Infant , Infant, Newborn , Male , Osteochondrodysplasias/genetics , Radiography , Syndrome
10.
Am J Med Genet ; 86(3): 269-73, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10482878

ABSTRACT

The oral-facial-digital syndrome type 1 (OFD1) includes limb, facial, intraoral malformations and the gene for the disorder was recently mapped to Xp22.3-p22.2. We report on monozygotic twin girls discordant for OFD1. Monozygosity is supported by placental pathology (monochorionic diamniotic) and molecular studies with probability of dizygosity <1 x 10(-6). The affected twin has oral cavity abnormalities including median cleft lip, cleft palate, lobulated hamartomatous tongue, aberrant hyperplastic oral frenula, alveolar notches, and absent lateral incisors. Facial manifestations include telecanthus, hypoplastic alae nasi, and transient neonatal facial milia. The patient also has short and deviated fingers with partial cutaneous syndactyly. At 10 years, she has not had central nervous system or kidney problems. X-inactivation study revealed similar X-inactivation patterns in the lymphoblasts of both twins. We conclude that skewed X-inactivation is an unlikely cause for the discordance, which is more likely due to a postzygotic mutation in the affected twin.


Subject(s)
Diseases in Twins/genetics , Orofaciodigital Syndromes/genetics , Child , Dosage Compensation, Genetic , Female , Humans , Mutation , Twins, Monozygotic
11.
J Clin Invest ; 101(9): 1881-8, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9576752

ABSTRACT

The GM2 gangliosidoses are a group of severe, neurodegenerative conditions that include Tay-Sachs disease, Sandhoff disease, and the GM2 activator deficiency. Bone marrow transplantation (BMT) was examined as a potential treatment for these disorders using a Sandhoff disease mouse model. BMT extended the life span of these mice from approximately 4.5 mo to up to 8 mo and slowed their neurologic deterioration. BMT also corrected biochemical deficiencies in somatic tissues as indicated by decreased excretion of urinary oligosaccharides, and lower glycolipid storage and increased levels of beta-hexosaminidase activity in visceral organs. Even with neurologic improvement, neither clear reduction of brain glycolipid storage nor improvement in neuronal pathology could be detected, suggesting a complex pathogenic mechanism. Histological analysis revealed beta-hexosaminidase-positive cells in the central nervous system and visceral organs with a concomitant reduction of colloidal iron-positive macrophages. These results may be important for the design of treatment approaches for the GM2 gangliosidoses.


Subject(s)
Bone Marrow Transplantation , Sandhoff Disease/therapy , beta-N-Acetylhexosaminidases/deficiency , Animals , Behavior, Animal , Brain Chemistry , Cerebral Cortex/pathology , Disease Models, Animal , Glycolipids/analysis , Longevity , Mice , Mice, Mutant Strains , Oligosaccharides/urine , Sandhoff Disease/mortality , Survival Analysis , beta-N-Acetylhexosaminidases/genetics
13.
Cleft Palate Craniofac J ; 34(5): 447-54, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9345615

ABSTRACT

OBJECTIVE: Infants born in Maryland between June 1992 and June 1996 were used in a case-control study of nonsyndromic oral clefts to test for effects of maternal smoking and a polymorphic genetic marker at the transforming growth factor alpha (TGFA) locus, both of which have been reported to be risk factors for these common birth defects. DESIGN AND SETTING: Cases were infants with an oral cleft ascertained through three comprehensive treatment centers, with additional ascertainment through a registry of birth defects maintained by the Maryland Health Department. Controls were healthy infants. Medical history information on infants and mothers were collected, along with DNA samples. PATIENTS, PARTICIPANTS: Among 286 cases contacted (72% ascertainment), there were 192 nonsyndromic isolated oral clefts (106 M; 86 F) available for this case-control study. MAIN OUTCOME MEASURES: The largest group of 149 Caucasian nonsyndromic cases and 86 controls was used to test for association with maternal smoking and genotype at the Taq1 polymorphism in TGFA. RESULTS: While this modest sample had limited statistical power to detect gene-environment interaction, there was a significant marginal increase in risk of having an oral cleft if the mother smoked (odds ratio = 1.75, 95% CI = 1.01 to 3.02). We could not demonstrate statistical interaction between maternal smoking and TGFA genotype in this study, however, and the observed increase in the C2 allele among cases was not statistically significant. CONCLUSIONS: We could not confirm either the reported association between oral clefts and TGFA genotype or its interaction with maternal smoking. However, these data do show an increased risk if the mother smoked during pregnancy, and this effect was greatest among infants with a bilateral cleft and no close family history of clefts.


Subject(s)
Cleft Lip/etiology , Cleft Palate/etiology , Pregnancy Complications , Smoking/adverse effects , Transforming Growth Factor alpha/genetics , Alleles , Case-Control Studies , Chromosome Mapping , Cleft Lip/genetics , Cleft Palate/genetics , DNA/genetics , Environment , Female , Gene Frequency , Genetic Markers , Genotype , Humans , Infant , Male , Maryland , Odds Ratio , Polymorphism, Genetic , Pregnancy , Registries , Risk Factors , Taq Polymerase/genetics
14.
Ann Med ; 29(6): 557-61, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9562524

ABSTRACT

Tay-Sachs disease and Sandhoff disease are severe neurodegenerative disorders caused by a deficiency of beta-hexosaminidase A and resultant accumulation of its substrate, GM2 ganglioside, in neuronal lysosomes. The three clinical forms of the disorders (infantile, juvenile and adult) are of varying severity and onset, and have been correlated with the amount of residual GM2 ganglioside-degrading activity present in patients' cells. Through targeted disruption of the murine beta-hexosaminidase genes in embryonic stem cells, we have developed a set of mice that vary in their GM2 ganglioside-degrading capacity and exhibit many of the clinical features of the human diseases. These mice are valuable for the study of pathogenic mechanisms and for devising novel therapeutic strategies in these disorders.


Subject(s)
Disease Models, Animal , Gangliosidoses/genetics , beta-N-Acetylhexosaminidases/deficiency , Adult , Animals , Child , G(M2) Ganglioside/metabolism , Gangliosidoses/therapy , Gene Targeting , Humans , Infant , Lysosomes/metabolism , Mice , Neurons/metabolism , Sandhoff Disease/genetics , Sandhoff Disease/therapy , Stem Cells/metabolism , Tay-Sachs Disease/genetics , Tay-Sachs Disease/therapy , beta-N-Acetylhexosaminidases/genetics
15.
Nat Genet ; 14(3): 348-52, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8896570

ABSTRACT

The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (alpha-subunit) and HEXB (beta-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, beta-hexosaminidase A (alpha beta), as well as for the homodimers beta-hexosaminidase B (beta beta) and S (alpha alpha). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs (Hexa-/-) and Sandhoff (Hexb-/-) disease model mice. Lacking both the alpha and beta-subunits these 'double knockout' mice displayed a total deficiency of all forms of lysosomal beta-hexosaminidase including the small amount of beta-hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients. This result demonstrates that glycosaminoglycans are crucial substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase enzyme system.


Subject(s)
Gangliosidoses/genetics , Lysosomes/enzymology , Mice, Knockout , Mucopolysaccharidoses/genetics , beta-N-Acetylhexosaminidases/genetics , Animals , Behavior, Animal , Brain/pathology , Chromatography, Thin Layer , Disease Models, Animal , Female , Fibroblasts/metabolism , Gangliosides/metabolism , Glycosaminoglycans/metabolism , Glycosaminoglycans/urine , Hexosaminidase A , Hexosaminidase B , Humans , Isoenzymes , Liver/enzymology , Male , Mice , Sphingolipids/chemistry , Sphingolipids/metabolism , Spleen/pathology
16.
J Biol Chem ; 271(29): 17377-82, 1996 Jul 19.
Article in English | MEDLINE | ID: mdl-8663217

ABSTRACT

The lysosomal beta-hexosaminidases are dimers composed of alpha and beta subunits. beta-Hexosaminidase A (alphabeta) is a heterodimer, whereas hexosaminidase B (betabeta) and S (alphaalpha) are homodimers. Although containing a high degree of amino acid identity, each subunit expresses a unique active site that can be distinguished by a differential ability to hydrolyze charged substrates. The site on the beta-subunit primarily degrades neutral substrates, whereas the alpha-subunit site is, in addition, active against sulfated substrates. Isozyme specificity is also exhibited with glycolipid substrates. Among human isozymes, only beta-hexosaminidase A together with the GM2 activator protein can degrade the natural substrate, GM2 ganglioside, at physiologically significant rates. To identify the domains of the human beta-hexosaminidase subunits that determine substrate specificity, we have generated chimeric subunits containing both alpha- and beta-subunit sequences. The chimeric constructs were expressed in HeLa cells to screen for activity and then selected constructs were produced in the baculovirus expression system to assess their ability to degrade GM2 ganglioside in the presence of GM2 activator protein. Generation of activity against the sulfated substrate required the substitution of two noncontinuous alpha-subunit sequences (amino acids 1-191 and 403-529) into analogous positions of the beta-subunit. Chimeric constructs containing only one of these regions linked to the beta-subunit sequence showed either neutral substrate activity only (amino acids 1-191) or lacked enzyme activity entirely (amino acids 403-529). Neither the chimeras nor the wild-type subunits displayed activator-dependent GM2-hydrolyzing activity when expressed alone. However, one chimeric subunit containing alpha amino acids 1-191 fused with beta amino acids 225 to 556, when co-expressed with the wild-type alpha-subunit, showed activity comparable with that of recombinant beta-hexosaminidase A formed by the co-expression of the alpha- and beta-subunits. This result indicates that the beta-subunit amino acids 225-556 contribute an essential function in the GM2-hydrolyzing activity of beta-hexosaminidase A.


Subject(s)
beta-N-Acetylhexosaminidases/chemistry , beta-N-Acetylhexosaminidases/metabolism , Amino Acid Sequence , Base Sequence , Carbohydrate Sequence , Cloning, Molecular , DNA Primers , G(M2) Ganglioside/chemistry , G(M2) Ganglioside/metabolism , Genetic Variation , HeLa Cells , Hexosaminidase B , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Substrate Specificity , Transfection
17.
Am J Med Genet ; 60(6): 535-40, 1995 Dec 18.
Article in English | MEDLINE | ID: mdl-8825891

ABSTRACT

We studied the neurodevelopmental profile of infants and toddlers with oculo-auriculo-vertebral spectrum (OAV) and determined if certain physical manifestations were indicative of a poor neurodevelopmental prognosis. Twenty-four patients with OAV, aged birth to 57 months, were seen in the Department of Medical Genetics at Children's National Medical Center for multidisciplinary evaluations, including neurodevelopmental assessments. Fifty-eight percent of these children scored more than 2 standard deviations below the mean in at least one domain of development. There was no difference in developmental outcome of boys versus girls, children affected unilaterally on the right side versus left side, and those with severe clinical manifestations versus those with a milder form. Children with OAV and abnormal muscle tone had lower cognitive, gross motor, and expressive language scores (P = 0.05, P = 0.002, and P = 0.02, respectively). Those affected bilaterally had lower cognitive, fine motor, receptive language, and expressive language scores (P = 0.06, P = 0.03, P = 0.03, P = 0.02, respectively). Children with cervical spine abnormalities had lower cognitive, fine motor, and expressive language scores (P = 0.02, P = 0.04, and P = 0.04, respectively). We conclude that infants and toddlers with OAV are at increased risk for neurodevelopmental delay, especially those with abnormal muscle tone, bilateral involvement, and cervical vertebral anomalies. The complexity of the neurodevelopmental problems is strongly suggestive of central nervous system disturbances. Patients with OAV need comprehensive evaluation by a multidisciplinary team to define potential neurodevelopmental delays, allow for early intervention services, and promote an optimal developmental outcome.


Subject(s)
Goldenhar Syndrome/physiopathology , Child, Preschool , Evoked Potentials, Auditory , Female , Humans , Infant , Intelligence Tests , Male , Motor Activity , Prognosis
18.
J Biol Chem ; 267(5): 3268-73, 1992 Feb 15.
Article in English | MEDLINE | ID: mdl-1737783

ABSTRACT

Human CD4, a monomeric T cell surface glycoprotein, is required for T helper cell activation and is also the receptor for the human immunodeficiency virus. There have been conflicting reports as to whether glycosylation of CD4 is required for its cell surface expression. To clarify the effect of glycosylation on surface expression, folding, and intracellular sorting of CD4, we generated a series of mutant cDNAs in which one, the other, or both glycosylation recognition sites were eliminated. Using in vitro transcription and translation we confirmed that both potential glycosylation sites of CD4 were utilized. Transient expression of the mutants in HeLa cells demonstrated that glycosylation at either site was necessary and sufficient for cell surface expression. Finally, we showed that unglycosylated CD4 produced in HeLa cells was incorrectly folded and retained intracellularly, probably in the endoplasmic reticulum.


Subject(s)
CD4 Antigens/metabolism , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Amino Acid Sequence , Antigens, CD/biosynthesis , Base Sequence , CD4 Antigens/genetics , Cell Membrane/immunology , Flow Cytometry , Fluorescent Antibody Technique , Glycosylation , HeLa Cells , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Protein Biosynthesis , Protein Conformation , T-Lymphocytes/physiology , Transcription, Genetic , Transfection
19.
Protein Expr Purif ; 1(2): 111-20, 1990 Nov.
Article in English | MEDLINE | ID: mdl-1967020

ABSTRACT

Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.


Subject(s)
Transferases (Other Substituted Phosphate Groups) , beta-N-Acetylhexosaminidases/biosynthesis , Baculoviridae/genetics , Cloning, Molecular , DNA/genetics , Humans , Lysosomes/enzymology , Mannosephosphates/chemistry , Molecular Weight , Phosphotransferases/metabolism , Protein Conformation , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Substrate Specificity , Trypsin , beta-N-Acetylhexosaminidases/genetics , beta-N-Acetylhexosaminidases/isolation & purification
20.
Am J Dis Child ; 142(7): 719-20, 1988 Jul.
Article in English | MEDLINE | ID: mdl-3381772

ABSTRACT

The records of 102 hospitalized preschool-age children were reviewed to determine their immunization status. Nineteen children (19%) had a documented delay in immunizations. None of these children were immunized prior to hospital discharge, although no contraindications to immunization had been identified. Two to five months after hospital discharge, ten patients (53%) were still deficient. Compliance with recommended immunization schedules is not adequately addressed during hospital admissions.


Subject(s)
Hospitalization , Immunization/statistics & numerical data , Child, Preschool , Humans , Infant , Maryland , Medical Records , Retrospective Studies
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