Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression.

Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic- and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and transphosphorylation of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals.

Two novel exonic point mutations in HEXA identified in a juvenile Tay-Sachs patient: role of alternative splicing and nonsense-mediated mRNA decay.

We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=), located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G (p.Asn399Asp) in exon 11, has been previously characterized as a common polymorphism in African-Americans. Hex A activity measured in TSD Glial cells, transfected with HEXA cDNA constructs bearing these mutations, was unaltered from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR products revealed three aberrant transcripts in the patient, one where exon 8 was absent, one where exon 11 was absent and a third lacking both exons 10 and 11. All three novel transcripts contain frameshifts resulting in premature termination codons (PTCs). Transfection of mini-gene constructs carrying the c.814G>A and c.1305C>T mutations proved that the two mutations result in exon skipping. mRNAs that harbor a PTC are detected and degraded by the nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal proteins. However, although NMD is functional in the patient's fibroblasts, aberrant transcripts are still present. We suggest that the level of correctly spliced transcripts as well as the efficiency in which NMD degrade the PTC-containing transcripts, apparently plays an important role in the phenotype severity of the unique patient and thus should be considered as a potential target for drug therapy.

Neurocognitive testing in late-onset Tay-Sachs disease: a pilot study.

OBJECTIVES: To test neurocognitive function in patients with late-onset Tay-Sachs disease (LOTS) using a computerized system to assess whether cognition is a clinically relevant outcome measure of possible therapeutic intervention in LOTS. METHODS: Ten adults with Tay-Sachs disease were administered at least one battery of the Mindstreams Neurotrax system for evaluation of cognitive function. Six sub-scores and a Global Cognitive Score (GCS) were tabulated. A disease specific severity score was also devised with six domains. RESULTS: Despite identical genotypes, all patients but the two oldest had > or = 3/6 sub-scores one standard deviation below normal mean (100); verbal and executive functions were most affected. The severity score measured other functions. CONCLUSIONS: Because of provocative findings on re-testing in patients exposed to miglustat, and despite the very small cohort, cognitive function may be an appropriate and clinically relevant outcome measure for future therapeutic interventions in LOTS.

Chromosome 10q harbors a susceptibility locus for bipolar disorder in Ashkenazi Jewish families.

We report the results of a 10 cM density genome-wide scan and further fine mapping of three chromosomal candidate regions in 10 Belgian multigenerational families with bipolar (BP) disorder. This two-stage approach revealed significant evidence for linkage on chromosome 10q21.3-10q22.3, showing a maximum multipoint parametric heterogeneity logarithm of odds (HLOD) score of 3.28 and a nonparametric linkage (NPL) score of 4.00. Most of the chromosome 10q evidence was derived from a single, large Ashkenazi Jewish pedigree. Haplotype analysis in this pedigree shows that the patients share a 14-marker haplotype, defining a chromosomal candidate region of 19.2 cM. This region was reported previously as a candidate region for BP disorder in several independent linkage analysis studies and in one large meta-analysis. It was also implicated in a linkage study on schizophrenia (SZ) in Ashkenazi Jewish families. Additionally, we found suggestive evidence for linkage on chromosome 19q13.2-13.4 (HLOD 2.01, NPL 1.09) and chromosome 7q21-q22 (HLOD 1.45, NPL 2.28). Together, these observations suggest that a gene located on chromosome 10q21.3-10q22.3 is underlying the susceptibility both for SZ and for BP disorder in at least the Ashkenazi Jewish population.

Is the WKL1 gene associated with schizophrenia?

A missense mutation Leu309Met in the WKL1 (MLC1, KIAA0027) gene, mapped to 22q13.3, was reported to co-segregate with periodic catatonic schizophrenia (SCZ) in a single large German pedigree with seven affected individuals (Meyer et al. [2001: Mol Psychiatry 6:302-306]). This report raised the following questions that were dealt with in the present study: does the Leu309Met mutation have a role in SCZ, or only in catatonic SCZ? Does the mutation Leu309Met in the WKL1 gene, encoding a putative membrane protein, non-selective cation channel, have any effect on the channel activity? Is the WKL1 gene, which is expressed exclusively in brain, expressed differently in SCZ brains compared to controls? These questions were answered by screening the Leu309Met mutation in 117 Israeli Jewish patients with SCZ (55 Ashkenazi and 62 non-Ashkenazi Jews) and 176 matched controls. In search of differences in the level of WKL1 gene expression, postmortem dorsalateral prefrontal cortex of 16 schizophrenic patients and 15 controls was checked. We also measured the putative channel activity of normal WKL1 subcloned in pcDNA3 to determine the effect of the reported Leu309Met mutation. Our results argue against the involvement of WKL1 in SCZ susceptibility.

31Phosphorus magnetic resonance spectroscopy in late-onset Tay-Sachs disease.

The late-onset form of GM2 gangliosidosis (Tay-Sachs disease) is an autosomal-recessive disorder with progressive neurologic disease, mainly characterized by motor neuron and spinocerebellar dysfunction. The majority of patients are of Ashkenazi Jewish origin. 31Phosphorus magnetic resonance spectroscopy of the brain was performed to study the metabolic changes of a 16-year-old patient with late-onset Tay-Sachs disease who had a heterozygous Gly269-->Ser mutation in the hexosaminidase A encoding gene in compound heterozygosity with another, yet unidentified mutation. Severe changes in phosphorus metabolism with a decreased amount of phosphodiesters and membrane-bound phosphates were demonstrated, suggesting an activation of phosphodiesterases by accumulating gangliosides. The clinical findings were well related to the changes in spectroscopically determined metabolites.

hKCa3/KCNN3 potassium channel gene: association of longer CAG repeats with schizophrenia in Israeli Ashkenazi Jews, expression in human tissues and localization to chromosome 1q21.

We demonstrate a significant association between longer CAG repeats in the hKCa3/KCNN3 calcium-activated potassium channel gene and schizophrenia in Israeli Ashkenazi Jews. We genotyped alleles from 84 Israeli Jewish patients with schizophrenia and from 102 matched controls. The overall allele frequency distribution is significantly different in patients vs controls (P = 0.00017, Wilcoxon Rank Sum test), with patients showing greater lengths of the CAG repeat. Northern blots reveal substantial levels of approximately 9 kb and approximately 13 kb hKCa3/KCNN3transcripts in brain, striated muscle, spleen and lymph nodes. Within the brain, hKCa3/KCNN3transcripts are most abundantly expressed in the substantia nigra, lesser amounts are detected in the basal ganglia, amygdala, hippocampus and subthalamic nuclei, while little is seen in the cerebral cortex, cerebellum and thalamus. In situ hybridization reveals abundant hKCa3/KCNN3 message localized within the substantia nigra and ventral tegmental area, and along the distributions of dopaminergic neurons from these regions into the nigrostriatal and mesolimbic pathways. FISH analysis shows that hKCa3/KCNN3 is located on chromosome 1q21.

At least six different mutations in HEXA gene cause Tay-Sachs disease among the Turkish population.

Twenty-five Turkish infants with Tay-Sachs disease (TSD) have been diagnosed in the past 8 years. All are from consanguineous, nonrelated families. The present study deals with the molecular basis of six Turkish TSD patients from five unrelated families in which the parents were first cousins. The five mutations identified in this study were INS-5 G-->A, R393X, R137X, 12-bp deletion in exon 10, and G454D. The first three were reported in earlier studies, two in Turkish TSD infants and one in a French TSD infant.

Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population.

Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that approximately 1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon.

Juvenile-onset spinal muscular atrophy caused by compound heterozygosity for mutations in the HEXA gene.

Progressive proximal muscle weakness is present both in spinal muscular atrophy (SMA) type III (Kugelberg-Welander disease) and in GM2 gangliosidosis, diseases that segregate in an autosomal recessive fashion. The SMN gene for SMA and the HEXA gene for GM2 gangliosidosis were investigated in a woman with progressive proximal muscle weakness, long believed to be SMA type III (Kugelberg-Welander type). She and her family underwent biochemical studies for GM2 gangliosidosis. Analysis of SMN excluded SMA. Biochemical studies on GM2 gangliosidosis showed deficiency in hexosaminidase A activity and increased GM2 ganglioside accumulation in the patient's fibroblasts. The HEXA gene was first analyzed for the Gly269-->Ser mutation characteristic for adult GM2 gangliosidosis. Since the patient was carrying the adult mutation heterozygously, all 14 exons and adjacent intron sequences were analyzed. A novel mutation in exon 1 resulting in an A-to-T change in the initiation codon (ATG to TTG) was identified. The adult patient is a compound heterozygote, with each allele containing a different mutation. Although mRNA was transcribed from the novel mutant allele, expression experiments showed no enzyme activity, suggesting that neither the TTG nor an alternative codon serve as an initiation codon in the HEXA gene.

[Juvenile GM2 gangliosidosis with progressive spinal muscular atrophy onset]. / Gangliosidose à GM2 juvénile débutant par une amyotrophie spinale progressive.

GM2 gangliosidosis are caused by a beta-hexosaminidase A enzyme deficiency. Mutations in the gene leaving residual enzyme activity give rise to juvenile and adult forms of the disease which have a great clinical heterogeneity. We report three cases which have been considered for some time as Kugelberg-Welander disease. beta-hexosaminidase A was determined with the sulfated synthetic substrate, 4-méthylumbelliferyl-N-acetylglucosamine 6-sulfate (4-MUGS), which allowed the diagnosis. Two of these cases from one family had normal values of hexosaminidase A in serum as found in the B1 variant. Compound mutations were detected. The B1 variants had a classical B1 mutation (G533-->A) and a new mutation located on exon 11. The patient of the second family had the classical mutation of adult GM2 gangliosidosis (Gly269-->Ser) and a new mutation on exon 1, at the initiation codon.

Molecular basis of heat labile hexosaminidase B among Jews and Arabs.

Genotyping individuals for Tay-Sachs disease (TSD) is mainly based on the heat lability of beta-hexosaminidase (Hex) A (alphabeta) and the heat stability of Hex B (betabeta). Mutations in the HEXB gene encoding the beta-subunits of Hex that result in heat-labile Hex B thus may lead to erroneous enzymatic genotyping regarding TSD. Utilizing single strand conformation polymorphism (SSCP) analysis for all 14 exons of HEXB followed by direct sequencing of aberrant fragments, we screened individuals whose Hex B was heat labile. A novel heat labile mutation, previously concluded to exist in the HEXB gene, was identified among Jews and Arabs as 1627 G-->A. One individual with heat labile Hex B (HLB) was negative for this novel mutation and for the known 1514 G-->A HLB mutation, proving that there exists at least one other unidentified HLB mutation. Based on these results, it is advisable to perform DNA tests for 1627 G-->A mutation in suspected HLB individuals.

Two mutated HEXA alleles in a Druze patient with late-infantile Tay-Sachs disease.

Two affected HEXA alleles were found in an Israeli Druze Tay-Sachs child born to first-cousin parents. His paternal allele contained two adjacent changes in exon 5: delta496C, which resulted in a frameshift and premature termination codon 96 nucleotides downstream, and 498C-->G, a silent mutation. The maternal allele had a 835T-->C transition in exon 8 (S279P). Phosphoimaging quantitation of the parents' RNAs showed that the steady-state levels of mRNAs of the mutant exons 5 and 8 were 5% and 50%, respectively, of normal levels. The exon 5 mutated allele with the premature translation termination resulted in severe deficiency of Hex A. Transient expression of the exon 8 mutated alpha-chain cDNA in COS-1 cells resulted in deficiency of enzymatic activity. The child exhibited a late-infantile-type disease.

Progressive cerebellar ataxia, proximal neurogenic weakness and ocular motor disturbances: hexosaminidase A deficiency with late clinical onset in four siblings.

Tay-Sachs disease is a genetically determined neurodegenerative disorder, resulting from mutations of the hexosaminidase (Hex) A gene coding for the alpha-subunit of beta-D-N-acetyl-hexosaminidase. Clinically, there is severe encephalomyelopathy leading to death within the first few years of life. Hex A activity is usually absent in tissue and body fluids of these patients. Juvenile and adult Hex A deficiencies are less severe but rare variants with some residual Hex A activity. All these variants are most prevalent among Ashkenazi Jews. We describe a non-Jewish family in which four adult brothers and sisters had markedly reduced Hex A activities and onset of symptoms in the second decade of life. The phenotypical expression was remarkably homogeneous, consisting in a combination of slowly progressive motor neuron disease, ataxia and ocular motor disturbances. None of the patients were demented at this stage of their illness. Magnetic resonance studies showed severe cerebellar atrophy, but were otherwise normal. Hex A deficiency was established by biochemical measurements in the serum and skin fibroblasts using the fluorogenic substrates 4-MUG and 4-MUGS as well as by gel electrophoresis. Molecular genetic studies revealed that the patients are compound heterozygotes for the 'adult' mutation Gly269 --> Ser and the 'infantile' 4-base insertion in exon 11 of the Hex A gene.

Novel HEXA mutation in a Bedouin Tay-Sachs patient associated with exon skipping and reduced transcript level.

An Israeli-Bedouin infant from a consanguineous family was diagnosed with Tay-Sachs disease (TSD). The patient was found to carry the novel +3tIVS4 mutation in homozygosity. Direct sequencing of the cDNA showed that the +3tIVS4 mutation caused complete skipping of exon 4 resulting in a stop codon 17 bp downstream. We postulate that the insertion disrupts base pairing between the consensus sequence and U1 snRNA causing exon skipping. The resultant transcript contains a premature termination codon and is severely reduced in amount. An extended nucleotide portion of the 5' end of IVS4 was sequenced.

Tay-Sachs disease and HEXA mutations among Moroccan Jews.

Moroccan Jewry (N>750,000) is the only non-Ashkenazi Jewish community in which Tay-Sachs disease (TSD) is not extremely rare. Previous studies among Moroccan Jewish TSD families identified three HEXA mutations. In this study, extended to enzyme-defined and new obilgate TSD carriers, we found four additional mutations. One of them is a novel, IVS5-2(A-->G) substitution, resulting in exon skipping, and it was found only among enzyme-defined carriers. The seven HEXA identified mutations among Moroccan Jews are: deltaF(304/305), R170Q, IVS-2(A-->G), Y180X, E482K, 1278+TATC, and IVS12+1(G-->C). Their respective distribution among 51 unrelated enzyme-defined and obligate carriers is 22:19:6:1:1:1:1. The mutation(s) remain unknown in only three enzyme-defined carriers. Five of the seven Moroccan mutations, including the three most common ones, were not found among Ashkenazi Jews. Compared with the much larger and relatively homogeneous Ashkenazi population, the finding among Moroccan Jews probably reflects their much longer history.

A new point mutation affecting the fourth transmembrane domain of PMP22 results in severe de novo Charcot-Marie-Tooth disease.

A novel T-->G mutation in exon 4 of the PMP22 gene was identified heterozygously in a girl with severe, de novo CMT1A disease. Duplication of the chromosomal 17p11-12 region, encompassing the PMP22 gene, was ruled out. This is the only known mutation that specifically affects the human fourth transmembrane (TM) domain of PMP22. It results in a substitution of a non-polar amino acid by a polar one (Leu147-->Arg), similar to the nearby Gly150-->Asp substitution, underlying the severe Trembler phenotype in the mouse. These mutations suggest that the fourth TM domain plays a crucial role in the normal function of PMP22. The new mutation also augments previous observations that diseases caused by mutations in PMP22 are more severe than those caused by the duplication of 17p11-12.

Vincristine treatment triggering the expression of asymptomatic Charcot-Marie-Tooth disease.

A 16-year-old male suffering from Ewing's sarcoma of the pelvis was treated with vincristine as part of his chemotherapeutic protocol. The boy was never known to suffer from any neurological problems. His father had a mild limp, attributed to prolonged "taxi driving," that was never investigated medically. The first course of treatment, which included 2 mg of vincristine, resulted in clinical improvement. However, at the same time the patient developed severe weakness of both upper and lower limbs, areflexia, and gradually a pes cavus deformity. Nerve conduction studies were suggestive of severe peripheral sensorimotor neuropathy, axonal and demyelinative. A definite diagnosis of Charcot-Marie-Tooth was confirmed by molecular analysis showing the typical duplication of 1.5 megabases at 17 p11.2. This unique manifestation of vincristine neurotoxicity is reported and discussed.