Mucolipidosis II and III. The genetic relationships between two disorders of lysosomal enzyme biosynthesis.

The genetic relationships between the multiple variants of mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy) were investigated with a sensitive genetic complementation analysis procedure. These clinically distinct disorders have defects in the synthesis of a recognition marker necessary for the intracellular transport of acid hydrolases into lysosomes. Both disorders are associated with an inherited deficiency of a uridine diphosphate-N-acetyl-glucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-phosphate transferase activity. We had previously shown that both disorders are genetically heterogeneous. Complementation analysis between mucolipidosis II and III fibroblasts indicated an identity of mucolipidosis II with one of the three mucolipidosis III complementation groups (ML IIIA), suggesting a close genetic relationship between these groups. The presence of several instances of complementation within this group suggested an intragenic complementation mechanism. Genetic complementation in heterokaryons resulted in increases in N-acetyl-glucosamine-phosphate transferase activity, as well as in the correction of lysosomal enzyme transport. This resulted in increases in the intracellular levels of several lysosomal enzymes and in the correction of the abnormal electrophoretic mobility pattern of intracellular beta-hexosaminidase. The findings demonstrate that a high degree of genetic heterogeneity exists within these disorders. N-acetyl-glucosamine-phosphate transferase is apparently a multicomponent enzyme with a key role in the biosynthesis and targeting of lysosomal enzymes.

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.

Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors.

Maternal side effects of prenatal dexamethasone therapy for fetal congenital adrenal hyperplasia.

UNLABELLED: Prenatal treatment of virilizing congenital adrenal hyperplasia in female fetuses via maternal dexamethasone (Dex) therapy (1-1.5 mg/day) from first trimester to birth was associated with excessive weight gain (47-56 pounds at 35-37 weeks gestation), Cushingoid facial features, severe striae resulting in permanent scarring, and hyperglycemic response (8-11.7 nmol/L) to oral glucose administration in all our experience (three cases). Other symptoms included hypertension, gastrointestinal intolerance, or extreme irritability. Previous pregnancies were uncomplicated by these problems. In each case, first or second trimester amniotic fluid 17-hydroxyprogesterone (17OHP, 17-41 nmol/L; normal less than 0.4 nmol/L), androstenedione (22-31 nmol/L, normal less than 5 nmol/L), and testosterone levels (0.54-0.7 nmol/L, normal less than 0.4 nmol/L) during Dex treatment were elevated regardless of the newborn genital outcome. Maternal serum estriol (E3) levels in one mother (normal newborn genitalia) were consistently low (less than 0.2 nmol/L) during the second and third trimester. In two mothers (partially virilized newborn genitalia) initial second trimester E3 levels were unsuppressed (11, 17.4 nmol/L) and suppressed (less than 1.4 nmol/L) following short-term increased dose. CONCLUSION: prenatal Dex treatment of virilizing congenital adrenal hyperplasia at a dose of 1-1.5 mg daily throughout gestation is associated with significant maternal side effects. Parents should be informed of these side effects before initiation of treatment. Careful monitoring for signs of side effects, medical intervention when necessary, and lowering of Dex dose during the second half of gestation would minimize the side effects. Maternal serum E3 levels appear useful for prediction of fetal outcome while amniotic fluid steroid levels may not.

Apparent allelism of the Hurler, Scheie, and Hurler/Scheie syndromes.

The Hurler, Scheie, and Hurler/Scheie syndromes are clinically distinct disorders due to inherited deficiencies of lysosomal alpha-iduronidase activity. The genetic relationship of these disorders was investigated by complementation analysis using a heterokaryon enrichment procedure. Fusions between fibroblasts derived from Hurler, Scheie, or Hurler/Scheie subjects in any combination resulted in no detectable correction of alpha-iduronidase activity, indicating that they form a single complementation group. Control fusions between these fibroblasts and mucolipidosis II or III cells, which are also alpha-iduronidase deficient due to an enzyme processing defect, resulted in restoration of up to 57% of normal alpha-iduronidase activity. These results also suggest allelism of the Hurler, Scheie, and Hurler/Scheie syndromes. However, the genetic make-up of the Hurler/Scheie syndrome was not resolved.

Fragile X syndrome: discordant levels of CGG repeat mosaicism in two brothers.

Fragile X syndrome is associated with an unstable repeated CGG trinucleotide sequence in the 5' untranslated region of the FMR-1 gene. A significant number of individuals with a mild or atypical presentation are mosaics for the CGG expansion. We report a family with two brothers. The proband had severe mental retardation as well as most of the Fragile X syndrome stigmata, whereas his brother shows only mild learning difficulties. Both inherited a 80 x CGG trinucleotide premutation from the mother. They were negative for the FRAXA fragile site in over 100 metaphases examined. Flanking markers verified that both had inherited the same FMR-1 allele and Xq27-q28 flanking sequences from the mother. The methylation status of the brothers indicated active FMR-1 transcription as determined by using StB12.3/EcoRI + Eagl blots. CGG size or methylation mosaicism was not apparent from Southern blots. Polymerase chain reaction and chemiluminescent detection identified that both brothers had different degrees of mosaicism for the CGG expansion. Large expansions amounting to 70% of the total were visible in the proband, whereas less than 5% of the signal was larger than the premutation in his mildly affected brother. These findings suggest that mosaicism may be responsible for some of the variation in penetrance in this disorder.

Genetic heterogeneity of I-cell disease is demonstrated by complementation of lysosomal enzyme processing mutants.

I-cell disease (mucolipidosis II) is a fatal childhood disorder affecting the expression of multiple lysosomal acid hydrolases. The disorder is characterized by clinical and biochemical heterogeneity which may reflect different mutants with a similar phenotype. Genetic complementation studies demonstrating genetic heterogeneity within this disorder are described utilizing cultured fibroblasts from 11 different patients. Fibroblasts from I-cell disease (ICD) and from five different lysosomal storage diseases with single structural gene enzyme deficiencies were fused in different combinations, and fractions enriched for multinucleated heterokaryons were isolated and tested for acid hydrolase activity and electrophoretic mobility. In fusions of ICD fibroblasts and various single lysosomal enzyme-deficient fibroblasts, the activity of the deficient enzyme and of the other ICD hydrolases were restored, demonstrating that ICD is not a lysosomal enzyme structural gene defect and that the ICD defect, and not just the single enzyme deficiency, is corrected. In fusions involving only I-cell fibroblasts, at least two complementation groups were identified by the recovery of activities of all lysosomal enzymes tested in heterokaryons. These results demonstrate the existence of genetic heterogeneity within the disorder and suggest that different mutations can result in the I-cell clinical and biochemical phenotype. The data support an altered post-translational processing of lysosomal enzymes as the cause of ICD and suggest that at least two genes participate in this pathway.

Uniparental isodisomy of chromosome 14 in two cases: an abnormal child and a normal adult.

Uniparental disomy (UPD) of a number of different chromosomes has been found in associated with abnormal phenotypes. A growing body of evidence for an imprinting effect involving chromosome 14 has been accumulating. We report on a case of paternal UPD of chromosome 14 studied in late gestation due to polyhydramnios and a ventral wall hernia. A prenatal karyotype documented a balanced Robertsonian 14:14 translocation. The baby was born prematurely with hairy forehead, retrognathia, mild puckering of the lips and finger contractures. Hypotonia has persisted since birth and at age one year, a tracheostomy for laryngomalacia and gastrostomy for feeding remain necessary. Absence of maternal VNTR polymorphisms and homozygosity of paternal polymorphisms using chromosome 14 specific probes at D14S22 and D14S13 loci indicated paternal uniparental isodisomy (pUPID). Parental chromosomes were normal. We also report on a case of maternal UPD in a normal patient with a balanced Robertsonian 14:14 translocation and a history of multiple miscarriages. Five previous reports of chromosome 14 UPD suggest that an adverse developmental effect may be more severe whenever the UPD is paternal in origin. This is the second reported patient with paternal UPD and the fifth reported with maternal UPD, and only few phenotypic similarities are apparent. Examination of these chromosome 14 UPD cases of maternal and paternal origin suggests that there are syndromic imprinting effects.

Three cell line mosaicism involving structural and numerical abnormalities of chromosome 18 in a 3.5-year-old girl: 47,XX,+18/47,XX,+del(18)(q22)/46,XX.

We report on a 3.5-year-old girl with a mosaic karyotype including full trisomy 18, normal cells and a majority of cells with partial trisomy involving an extra chromosome 18 deleted at band q22. She had cardiac and CNS anomalies, dysmorphic facial features failure to thrive and developmental delay. A gastrostomy tube was placed at 2 years of age. The combination of improved nutrition and optimal developmental therapy has led to her sitting supported, attempting to stand and enhancement of her cognitive and non-verbal communication abilities. Molecular investigation of the patient and her parents using microsatellite analysis has led to the conclusion that, as expected, the additional copy of chromosome 18 constituting the full trisomic cell line is maternal meiosis I in origin. The data, however, indicate that in the trisomic cell line containing the deleted chromosome 18q, the structurally abnormal 18 was of paternal origin. We think this case is the first described with both structural and numerical trisomic mosaicism involving chromosome 18 in a liveborn infant. We propose a mechanism of origin and review the literature, comparing the clinical presentation of this case with individuals having full or partial trisomy 18.

Mucolipidosis I: studies of sialidase activity and a prenatal diagnosis.

The characteristics of the sialidase (N-acetyl-alpha-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[3H]lactitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid, or 2'-(4-methylumbelliferyl)-N-acetyl-alpha-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and beta-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[3H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell cultures.

I-cell disease and pseudo-Hurler polydystrophy: heterozygote detection and characteristics of the altered N-acetyl-glucosamine-phosphotransferase in genetic variants.

The human disorders I-cell disease and pseudo-Hurler polydystrophy (also known as mucolipidosis II and III, respectively) are caused by an inherited deficiency of UDP-GlcNAc: lysosomal enzyme precursor GlcNAc-P transferase activity. The most common genetic variants of these diseases (complementation group A) can be identified in homozygotes and heterozygotes using a GlcNAc-P transferase assay with artificial acceptors and commercially available radiochemicals. The kinetic characteristics of the residual GlcNAc-P transferase activity in complementation group A fibroblasts indicates that the low activity is due to a low Vmax. The measured Michaelis-Menten constants for the substrates UDP-GlcNAc and alpha-methyl mannoside are in the normal range. Homozygotes and heterozygotes of another less common variant of pseudo-Hurler polydystrophy (complementation group C) have normal activity and normal kinetic characteristics with this assay using alpha-methyl mannoside as the acceptor substrate. Several PHP variants with unusual characteristics are discussed.

Twin-twin transfusion syndrome in a dizygotic monochorionic-diamniotic twin pregnancy.

We present a case of twin-twin transfusion syndrome with discordant gender. Monochorionicity was confirmed by surgical pathology. Cytogenetic analysis showed normal 46,XX and 46,XY karyotypes. Microsatellite analysis using reliable pericentromeric markers was consistent with dispermic fertilization of two separate ova. This suggests that monochorionicity, rather than zygosity, may be responsible for the development of placental vascular anastomoses.

Peripheral neuropathy in the first 2 years of life: diagnostic evaluation including molecular genetics.

Inherited polyneuropathies present in the first 2 years of life are discussed with emphasis on clinical, pathologic, and molecular data. Early-onset polyneuropathies are relatively rare, sometimes life-threatening conditions that demand early recognition by clinical and pathologic examination. Histologic and ultrastructural overlaps among the various conditions are sometimes resolved by molecular genetic analysis. The growth in disease identification by genetic localization allows a more comprehensive understanding of the clinical and morphologic heterogeneity involving rearrangements of the same gene. Molecular mechanisms explaining the acquisition of such gene rearrangements are beginning to be unraveled. Peripheral myelin disorders may be confused with primary axonal disorders, and electrophysiologic examination often helps to distinguish between these two. Furthermore, early-onset central nervous system disorders may present as peripheral polyneuropathies and confound the clinical picture. A tentative diagnosis can often be offered by pathologic examination and confirmed by biochemical enzyme analysis later. The differential clinical diagnostic considerations of early-onset polyneuropathies are offered, to help clinicians sort out these diseases in the most efficient manner.

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis: genetic complementation analysis of the neuraminidase deficiency.

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis [ML(gal-neur-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal-neur-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular beta-galactosidase and alpha-neuraminidase levels in heterokaryons. The lack of human beta-galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal-neur-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect mat occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal beta-galactosidase and alpha-neuraminidase.

beta-Glucoside hydrolase activity of normal and glucosylceramidotic cultured human skin fibroblasts.

Cultured human skin fibroblasts from normal and glucosylceramidotic subjects are found to contain one beta-glucoside hydrolase as compared with multiple enzymes in other tissues. The fibroblast enzyme has an approximate molecular weight of 150,000 under isotonic conditions, as determined by gel filtration. It occurs as a large aggregate at low ionic strength. Ceramide, 4-methylumbelliferyl, and p-nitrophenyl beta-glucosides are active as substrates. The enzyme in whole cell homogenates is membrane-bound and is solubilized by a combination of Triton X-100 and sodium taurocholate. It has a pH optimum at 4.2 and no demonstrable divalent cation requirement. The cultured fibroblast beta-glucosidase displays close similarity to one of the forms of beta-glucosidase in human spleen, specifically that form which is affected in Gaucher's disease. 4-Methylumbelliferyl beta-glucosidase activity in homozygous fibroblasts from infantile and adult forms of Gaucher's disease are reduced to 9 and 14%, respectively, of normal fibroblast activity. The residual activity in the lipidotic cells shows increased heat lability, but cannot be distinguished from that in normal cells with respect to gel exclusion properties, Michaelis constant, and pH dependence.

Presymptomatic testing for Huntington's disease. Emotional aspects.

Presymptomatic genetic testing for Huntington's disease has been available since 1986. Recent discovery of the distinct gene by the Huntington's Disease Collaborative Research Group will probably simplify testing and encourage more people to pursue it. Unfortunately advances in treatment lag behind genetics diagnosis capabilities. The individual at risk faces pursuing a test for a disease for which there is no cure, only limited treatment. The emotional aspects are reviewed of individuals being tested at the Regional Genetic Program at the University of South Florida from May 1988 until April 1993.

UDP-N-acetylglucosamine: lysosomal enzyme precursor N-acetylglucosamine-1-phosphate transferase activities in human ovarian tumor tissue and some transformed cell lines.

Uridine diphosphate-N-acetylglucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-1-phosphate transferase, is a key enzyme involved in the intracellular targeting of lysosomal enzymes. This enzyme is elevated fourfold in primary ovarian tumor microsomes with respect to normal ovarian microsomes. This elevation is associated with significant increases in the specific activity of multiple lysosomal hydrolases, including beta-D-hexasaminidase, alpha-L-fucosidase, and beta-D-galactosidase. The activity of the phosphotransferase was also documented in several cell lines derived from human tumors. The possible role of this enzyme in tumor-associated phosphorylation is discussed.

Cell line segregation in a 45,X/46,XY mosaic child with asymmetric leg growth.

Clinical evaluation of a 13 1/2-year-old male revealed a 4.4-cm leg length discrepancy and a small penis with a normal endocrine evaluation. Cytogenetic analysis of peripheral blood lymphocytes and skin fibroblasts derived from the back showed 45,X/46,XY mosaicism with similar percentages of 45,X cells, 36% and 30% respectively. However, two separate skin fibroblast cultures derived from the thigh and calf of the short (right) leg showed significant lack of Y-bearing cells with 100% and 80% 45,X, respectively. In contrast, skin biopsies of the thigh and calf of the normal (left) leg both showed 100% 46,XY. Similar evidence for differences in the percentages of Y-bearing cells in the left versus right leg fibroblast cultures was obtained using densitometric scanning of dot blots following DNA hybridization with a Y-specific probe at the DYZ4 locus. Asymmetric limb growth in cases of X/XY lymphocyte mosaicism warrants further cytogenetic investigation to substantiate possible genotype-phenotype correlations which may help uncover the fundamental growth deficiency in Turner syndrome.

Correction of mucolipidosis III in vitro by gene transfer.

Mucolipidosis II (ML II, I-cell disease) and mucolipidosis III (ML III, pseudo-Hurler polydystrophy) are human autosomal recessive genetic disorders resulting from deficient UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1-phosphotransferase (GNPT) activity. Normally, this enzyme is involved in the processing of most lysosomal enzymes. Cultured fibroblasts from individuals with either disorder are deficient in a broad array of lysosomal enzymes as a result of the diminished GNPT activity. We report the correction of this phenotype by fusing transformed ML III cells generated for this study to lethally irradiated rodent cells. This method of gene transfer does not require selection for the gene of interest, animal models, nor any knowledge of the gene product except a screening method for its presence. It has generated corrected cell hybrids that contain approximately 1% hamster-derived sequences. These cell lines, which contain the hamster analogue to the human phosphotransferase gene, are useful for the molecular cloning of the gene defective in ML II and ML III.