Identification of the gene causing mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12?15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.

On the uptake of materials by the intact liver. The concentrative transport of rubidium-86.

In this study we use the multiple indicator dilution technique to outline the kinetic mechanisms underlying the uptake of rubidium, a cation which, in the steady state, is concentrated by hepatic parenchymal cells. We inject a mixture of (51)Cr-labeled red blood cells (a vascular reference substance), (22)Na (which is confined to the extracellular space, the expected extravascular distribution space for rubidium, in the absence of cellular uptake), and (86)Rb into the portal vein and obtain normalized outflow patterns, expressed as outflowing fractions of each injected mass per milliliter vs. time. The labeled red cell curve rises to the highest and earliest peak and decays rapidly. That for labeled sodium rises to a later and lower peak, and decays less rapidly. Its extrapolated recovery is equal to that for the red cells. The observed (86)Rb curve consists of two parts: an early clearly defined peak of reduced area, related to the (22)Na peak in timing; and a later tailing, obscured by recirculation, so that total outflow recovery cannot be defined (even though it would be expected to be the same). We model the concentrative uptake of (86)Rb and find two corresponding outflow fractions: throughput material, which sweeps past the cell surface as a wave delayed with respect to the vascular reference (tracer which has not entered cells); and exchanging material (tracer which has entered cells and later returns to the circulation). We find that the outflow form of the rubidium curve, the presence of both a relatively clearly defined throughput component and a relatively prolonged low-in-magnitude tailing, is consequent to the concentrative character of the transport mechanism, to the presence of an influx rate constant many times the efflux rate constant. The modeling which we develop is general, and has potential application in situations where transport is nonconcentrative.

On the uptake of materials by the intact liver. The transport and net removal of galactose.

D-galactose, a monosaccharide rapidly phosphorylated within liver cells, is irreversibly removed from the portal circulation. We have studied the kinetic relations between the hepatic cell entry process and the metabolic sequestration process, by means of the multiple indicator dilution technique. Labeled red blood cells (a vascular indicator), labeled sucrose (an extracellular reference), and labeled galactose were rapidly injected into the portal vein, and from rapidly sampled hepatic venous blood, normalized outflow-time patterns were secured. The labeled red cell curve rises to the highest and earliest peak, and decays rapidly; and that for labeled sucrose rises to a later and lower peak. Its extrapolated recovery is equivalent to that of the labeled red cells. At low blood galactose concentrations, the labeled galactose appears at the outflow with labeled sucrose, but is much reduced in magnitude, and exhibits a long tailing. Its outflow recovery is much reduced. At high blood galactose concentrations, the initial part of the profile increases towards that for labeled sucrose, the tailing becomes much larger in magnitude, and the outflow recovery becomes virtually complete. We have modeled the uptake of labeled galactose, and find two parts to the predicted outflow pattern, corresponding to our experimental observations; throughput material, which sweeps past the cell surface in the extracellular space; and returning material, which has entered the cells but escaped the sequestration process. Analysis of the data by use of this model provides estimates of both transmembrane fluxes and rates of sequestration. The capacity of the process subserving cell entry is found to be 40 times that for phosphorylation; and, whereas the K(m) value for sequestration is less than 15 mg/100 ml, that for entry is approximately 500 mg/100 ml. Both processes are relatively stereospecific; the entry of the L-stereoisomer is very slow and it undergoes no significant amount of metabolic sequestration. The sequestration process produces a lobular intracellular concentration gradient; and this gradient, in turn, produces some uncertainty in the estimate of the true K(m) value for the sequestration process.

Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and are paradoxically enlarged with dietary zinc deficiency.

The ZnT3 zinc transporter is uniquely expressed in cortical glutamatergic synapses where it organizes zinc release into the synaptic cleft and mediates beta-amyloid deposition in transgenic mice. We studied the association of zinc in plaques in relation to cytoarchitectural zinc localization in the APP/PS1 transgenic mouse model of Alzheimer's disease. The effects of low dietary zinc for 3 months upon brain pathology were also studied. We determined that synaptic zinc distribution within cortical layers is paralleled by amyloid burden, which is heaviest for both in layers 2-3 and 5. ZnT3 immunoreactivity is prominent in dystrophic neurites within amyloid plaques. Low dietary zinc caused a significant 25% increase in total plaque volume in Alzheimer's mice using stereological measures. The level of oxidized proteins in brain tissue did not changed in animals on a zinc-deficient diet compared with controls. No obvious changes were observed in the autometallographic pattern of zinc-enriched terminals in the neocortex or in the expression levels of zinc transporters, zinc importers or metallothioneins. A small decrease in plasma zinc induced by the low-zinc diet was consistent with the subclinical zinc deficiency that is common in older human populations. While the mechanism remains uncertain, our findings indicate that subclinical zinc deficiency may be a risk factor for Alzheimer's pathology.

Cognitive behavioral therapy for compulsive buying behavior: Predictors of treatment outcome.

BACKGROUND: Compulsive buying behavior (CBB) is receiving increasing consideration in both consumer and psychiatric-epidemiological research, yet empirical evidence on treatment interventions is scarce and mostly from small homogeneous clinical samples. OBJECTIVES: To estimate the short-term effectiveness of a standardized, individual cognitive behavioral therapy intervention (CBT) in a sample of n=97 treatment-seeking patients diagnosed with CBB, and to identify the most relevant predictors of therapy outcome. METHOD: The intervention consisted of 12 individual CBT weekly sessions, lasting approximately 45minutes each. Data on patients' personality traits, psychopathology, sociodemographic factors, and compulsive buying behavior were used in our analysis. RESULTS: The risk (cumulative incidence) of poor adherence to the CBT program was 27.8%. The presence of relapses during the CBT program was 47.4% and the dropout rate was 46.4%. Significant predictors of poor therapy adherence were being male, high levels of depression and obsessive-compulsive symptoms, low anxiety levels, high persistence, high harm avoidance and low self-transcendence. CONCLUSION: Cognitive behavioral models show promise in treating CBB, however future interventions for CBB should be designed via a multidimensional approach in which patients' sex, comorbid symptom levels and the personality-trait profiles play a central role.

Subjective craving and event-related brain response to olfactory and visual chocolate cues in binge-eating and healthy individuals.

High-sugar/high-fat foods are related to binge-eating behaviour and especially people with low inhibitory control may encounter elevated difficulties to resist their intake. Incentive sensitization to food-related cues might lead to increased motivated attention towards these stimuli and to cue-induced craving. To investigate the combined influence of olfactory and visual stimuli on craving, inhibitory control and motivated attention, 20 healthy controls and 19 individuals with binge-eating viewed chocolate and neutral pictures, primed by chocolate or neutral odours. Subjective craving and electroencephalogram activity were recorded during the task. N2 and Late Positive Potential (LPP) amplitudes were analysed. Patients reported higher craving than controls. Subjective craving, N2 and LPP amplitudes were higher for chocolate versus neutral pictures. Patients showed a higher relative increase in N2 amplitudes to chocolate versus neutral pictures than controls. Chocolate images induced significant increases in craving, motivated attention and measures of cognitive control. Chocolate odour might potentiate the craving response to visual stimuli, especially in patients with binge-eating.

Inhibition of protein kinase C activity in mucolipidosis type-4: a model for a new pathogenetic mechanism in inborn errors of metabolism.

Inhibition of protein kinase C [PK-C] activity by sphingosine and its derivatives has been suggested to play a role in the pathogenesis of sphingolipidoses. In the present study, PK-C activity and PK-C-mediated phosphorylation of endogenous substrates were studied in skin fibroblasts from patients with mucolipidosis type 4 [ML-4], in which there is accumulation of the phospholipids phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine as well as gangliosides. Cytosolic PK-C activity in 5 ML-4 cell lines was comparable to that in control cells. PK-C activity in the particulate fraction of these cells was 84 +/- 14 pmol 32P/mg protein per min compared with 267 +/- 26 in control cells. Increasing the concentrations of the activating lipids in the reaction mixture did not enhance PK-C activity in ML-4 cells, suggesting a non-competitive inhibition of the kinase. Following partial purification of the enzyme from the particulate fraction PK-C activity increased to 288 +/- 14 and 339 +/- 12 pmol 32P/mg protein per min in ML-4 and control cells, respectively. The phosphorylation pattern of endogenous substrates in the particulate fraction of ML-4 cells differed from that in control cells both in the absence and in the presence of calcium and activating lipids. We suggest that PK-C may be involved in the pathogenesis of sphingolipidoses and that this may represent an example for a new type of pathogenetic mechanisms in inborn errors of metabolism.

Mucolipidosis type IV: novel MCOLN1 mutations in Jewish and non-Jewish patients and the frequency of the disease in the Ashkenazi Jewish population.

The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.

Mucolipidosis type IV: the origin of the disease in the Ashkenazi Jewish population.

Mucolipidosis type IV (MLIV) is a neurodegenerative lysosomal storage disease in which most of the patients diagnosed hitherto are Ashkenazi Jews. The basic metabolic defect causing this disease is still unknown and the relevant gene has not yet been mapped or cloned. Seventeen Israel Ashkenazi families with MLIV patients had been interviewed to study their family origin. Although the families immigrated to Israel from various European countries they all could trace their roots three to four generations back to northern Poland or the immediate neighbouring country, Lithuania. Furthermore, there are only one or two ultraorthodox families among the 70-80 Ashkenazi families with MLIV patients worldwide, a marked under-representation of this group which constitutes at least 10% of the Ashkenazi population. This data indicate that MLIV mutation occurred only around the 18th and 19th centuries, after the major expansion of this population, in a founder in this defined European region belonging to a more modern, secular family.

Mucolipidosis type IV: clinical spectrum and natural history.

The clinical spectrum and developmental features of mucolipidosis type IV, a recessive lysosomal storage disorder, are presented. The evaluation was based on information from the clinical charts and information obtained from the families of 20 patients between the ages of 2 to 17 years. The clinical manifestations of the disease, profound psychomotor retardation and visual impairment, appear during the first year of life. Definitive diagnosis is made by electron microscopy which reveals storage organelles typical of the mucolipidoses. This study details, for the first time, the heterogeneity of the ophthalmologic features, specifically as pertains to the age of onset, degree and clinical course of the corneal opacities, and the retinal involvement. Although the top developmental level was found to be 12 to 15 months in language and motor function, the course of the disease is protracted for some children, who show only a slight improvement, and others, little if any deterioration despite the early infantile onset of the disease. This presentation provides guidelines for the clinical diagnosis of mucolipidosis type IV.

Deficiency of lysosomal hydrolases in apparently healthy individuals.

The deficiency of a lysosomal hydrolase usually results in the storage of its substrate(s) leading to various clinical abnormalities, typical for each deficiency. However, in certain lysosomal hydrolases, an apparent deficiency was noted which does not result in the classical clinical picture. This condition was described for aryl sulfatase A, beta-hexosaminidase, alpha-galactosidase, and galactocerebrosidase, where apparently healthy individuals showed in vitro very low hydrolase activity, usually indistinguishable from the affected patients. The deficiency was usually observed with both the synthetic and natural substrates. In the case of aryl sulfatase A deficiency, no clinical abnormalities were noted in these individuals, and cultured cells obtained from them were able to catabolize normally the natural substrate. Such cases are therefore referred as pseudodeficient. In other cases, such as in beta-hexosaminidase-A deficiency, mild manifestations of the corresponding disorder were reported with subsequent intralysosomal storage of GM2 ganglioside. Our analysis indicates that most of these cases represent a compound heterozygote for the deficient allele and another allele coding for an in vitro low enzyme activity (pseudodeficiency). A complete biochemical explanation for this phenomena is not yet established. The importance of understanding this condition(s) for proper genetic counseling is discussed.

Heterozygote detection in Hunter syndrome.

Iduronate sulfate sulfatase activity was determined in 36 women, relatives of Hunter syndrome patients. The use of serum and lymphocyte extracts for the determination of enzyme levels enabled the detection of 13 out of 15 (86%) obligate heterozygotes and identification of 10 of 21 other relatives as carriers. These methods are relatively simple and can easily be applied for routine examinations of all women at risk of being a Hunter heterozygote. These results permit for the first time meaningful genetic counseling for the families of Hunter patients.

Screening for carriers of Tay-Sachs disease in the ultraorthodox Ashkenazi Jewish community in Israel.

A screening program for the detection of Tay-Sachs disease (TSD) carriers in the ultra Orthodox community of Ashkenazi Jews has operated in Israel since 1986. The purpose of this program is the prevention of marriages of 2 heterozygotes. The screened individuals are mostly couples in the engagement process or students in religious high schools. Two mandatory requirements guide this program. First, anonymity of the tested individuals who are identified only by code numbers; second completion of the test results of couples in the engagement process within a few days. The screening program is performed by the determination of hexosaminidase A (Hex A) activity in serum which is repeated in serum and leukocyte extracts in couples where both partners were found in the heterozygote range in the initial tests. The minimal carrier frequency was estimated to be 1:26 or higher, which is higher then in the general Jewish Ashkenazi population. This higher carrier frequency apparently stems from the fact that most members of this community originate from central Europe where the TSD carrier frequency was previously reported to be the highest in the Ashkenazi population. Since the beginning of the screening program no TSD child has been born to newlywed couples of this community in Israel.

Tay-Sachs screening in the Jewish Ashkenazi population: DNA testing is the preferred procedure.

A unique screening program for the identification of Tay-Sachs Disease (TSD) heterozygotes has been performed in the tradi- tional Orthodox Ashkenazi Jewish (AJ) community since 1983. In recent years the program has utilized the biochemical assay for the determination of hexosaminidase A levels by the heat inactivation technique as well as by direct DNA analysis. The three mutations which were analyzed were those that have been shown to be prevalent among AJ TSD patients and carriers, namely the four nucleotide insertion mutation in exon 11 (1278+TATC), the splice mutation at the 5' end of intron 12 (1421+1g-->c), and the adult mutation, a Gly(269)-->Ser substitution in exon 5 (G269S). A total of 103,133 individuals were tested by biochemical analysis, and 38,197 of them were also assayed by DNA testing. Furthermore, 151 chromosomes from TSD patients or obligate heterozygotes were subjected to DNA analysis for one of the three mutations. DNA testing of the latter identified one of the three AJ mutations in every case, predicting a very high detection rate of heterozygotes in this community by this method. By contrast, the sensitivity of the enzyme assay ranged from 93.1% to 99.1% depending on the exclusion (inclusion) of inconclusive results as positive, while the specificity ranged from 88.1% to 98.8% depending on the inclusion (exclusion) of inconclusive results as positive. Our results strongly support the use of DNA testing alone as the most cost-effective and efficient approach to carrier screening for TSD in individuals of confirmed Ashkenazi Jewish ancestry.

Gaucher disease type I and pregnancy.

We surveyed 47 pregnancies of 17 women affected with Gaucher disease (GD) type I. In two women affected with the severe form of GD type I, no change was observed in the course of the disease during pregnancy. In one patient with the moderate form of the disease there was an exacerbation of the disease during and after pregnancy, and thereafter two subsequent pregnancies of this woman ended by early spontaneous abortion. Four women were diagnosed during their pregnancy or soon after delivery suggesting in these women an exacerbation related to pregnancy. In the other ten women there was no change in the course of the disease. In general, the pregnancies of women affected with GD were normal; however, six women needed blood transfusion during pregnancy or at delivery. From these data it is suggested that there is some risk to pregnant women affected with GD type I, and accordingly, appropriate follow-up should be planned at the beginning of pregnancy in these patients.

Krabbe disease: increased incidence in a highly inbred community.

Krabbe disease (globoid cell leukodystrophy) was found with very high incidence (6/1,000 live births) in a large Druze kindred in Israel. The clinical data on 12 of the affected children demonstrated clinical variability even though these children are homozygous for the same mutation by descent from a common ancestor.

Combined enzymatic and linkage analysis for heterozygote detection in Hunter syndrome: identification of an apparent case of germinal mosaicism.

Hunter syndrome is an X-linked recessive disorder. Determination of heterozygotes is of vital importance in genetic counselling. We describe the DNA linkage analysis in 6 Hunter syndrome families and compare it to previous results based on a serum assay for IDS activity. Our results confirm the reliability of the serum assay. The serum test correctly detected 11/12 of the 1st degree relatives tested by the serum assay (6/7 carriers and 5/5 non-carriers). The only case with an apparent false negative result in the serum test was a daughter of a "probable heterozygote" whose serum test was also negative. We suggest that in this family the mother represented a case of germinal mosaicism and her daughter, based on the serum test, was not a carrier. If our interpretation is correct, then the apparent false negative results were correct. It is concluded that in families where the mutation is not known and DNA analysis is not possible due to the lack of informative RFLPs or due to the lack of DNA samples on key individuals, as well as in sporadic cases, the serum test should be applied as an alternative option for heterozygote detection.

Aryl sulfatase A deficiency in psychiatric and neurologic patients.

Two hundred ninety-five psychiatric and neurologic patients were randomly screened for aryl sulfatase A (ASA) activity in lymphocyte extracts. Two of these patients showed very low ASA activity, in the range of metachromatic leukodystrophy (MLD)-affected patients. The residual activity in these low ASA patients showed normal enzyme behavior with regard to ASA kinetic features and the ability to catabolize 14C labeled sulfatide by intact fibroblasts. Taking into account that approximately 3% of the general population are homozygous for the pseudo-aryl sulfatase A gene and are clinically unaffected, the data obtained here indicate that the patients studied in this work, as well as most psychiatric patients reported in the literature with low ASA activity, represent the normal ASA polymorphism. Thus, the very low ASA activity patients are in fact homozygous for the pseudo-deficient allele, which does not result in clinical abnormalities. The clinical symptoms in these psychiatric patients and probably other "variant" MLD patients are therefore not related to low ASA activity.

Mucolipidosis type IV: a mild form with late onset.

A 16-year-old girl is presented with mild clinical manifestations and late onset of mucolipidosis type IV (MLIV). The patient, an Ashkenazi Jew, has had minor motor difficulties and mild psychological disturbances since early childhood. Her vision began deteriorating at 12 years of age, due to bilateral corneal opacities and retinal degeneration. At present she attends a regular high school, although she is slow and scholastic achievements are lower than average. Electron microscopic examination and biochemical studies were typical for MLIV, namely, abnormal ganglioside retention and typical pattern of phospholipids accumulation. This very mild presentation of MLIV suggests a broader spectrum of heterogeneity of this disorder and raises the possibility that MLIV, at least among Ashkenazi Jews, might be more frequent than estimated hitherto, due to undiagnosed mild patients.

Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise.

We examined exercise-induced changes in indicator-dilution estimates of the angiotensin-converting enzyme first-order kinetic parameter, the ratio of a normalized maximal enzymatic conversion rate to the Michaelis constant (Amax/Km), which, under stable enzymatic conditions, will vary with the pulmonary vascular surface area accessible to vascular substrate, the extravascular lung water (an index of the proportion of lung tissue perfused), and the central blood volume (from pulmonary trunk to aorta). Experiments were performed in 10 mongrel dogs at rest and through two increasing levels of treadmill exercise, with the use of two vascular space tracers (labeled erythrocytes and albumin), a water space tracer ([1,8-14C]-octanediol), and a vascular endothelium surface area marker, benzoyl-Phe-Gly-Pro ([3H]BPGP), which is a pharmacologically inactive angiotensin-converting enzyme substrate. The exercise-induced increase in cardiac output was accompanied by a linear increase in central blood volume, and dilutional extravascular lung water rapidly increased to an asymptotic proportion close to 100% of postmortem vascular lung water. There was an average 55% [3H]BPGP hydrolysis, which did not vary with flow, and the computed Amax/Km increased linearly with exercise. We conclude that exercise results in complete lung tissue recruitment and increases the pulmonary vascular surface area available for BPGP hydrolysis linearly with flow, so that pulmonary vascular recruitment continues after full tissue recruitment.