Mutation in CUL4B, which encodes a member of cullin-RING ubiquitin ligase complex, causes X-linked mental retardation

We reevaluated a previously reported family with an X-linked mental retardation syndrome and attempted to identify the underlying genetic defect. Screening of candidate genes in a 10-Mb region on Xq25 implicated CUL4B as the causative gene. CUL4B encodes a scaffold protein that organizes a cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. A base substitution, c.1564C-->T, converted a codon for arginine into a premature termination codon, p.R388X, and rendered the truncated peptide completely devoid of the C-terminal catalytic domain. The nonsense mutation also results in nonsense-mediated mRNA decay in patients. In peripheral leukocytes of obligate carriers, a strong selection against cells expressing the mutant allele results in an extremely skewed X-chromosome inactivation pattern. Our findings point to the functional significance of CUL4B in cognition and in other aspects of human development.     

Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus

We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes.     

Linkage mapping of a severe X-linked mental retardation syndrome.

A four-generation Swedish family with a new type of X-linked mental retardation syndrome was recently reported by Gustavson et al. The complex syndrome includes microcephaly, severe mental retardation, optical atrophy with decreased vision or blindness, severe hearing defect, characteristic facial features, spasticity, seizures, and restricted joint motility. The patients die during infancy or early in childhood. Twenty-one family members, including two affected males, were available for study. Linkage analysis was conducted in the family by using 11 RFLP markers and 10 VNTR markers spread along the X chromosome. A hypervariable short tandem repeat of DXS294 at Xq26 showed a peak two-point lod score of 3.35 at zero recombination fraction. Calculations using the same markers revealed a multipoint peak lod score of 3.65 at DXS294. Crossover events with the centromeric marker DXS424 and the telomeric marker DXS297 delimit a probable region for the gene localization. It is noteworthy that hte disease loci of two other syndromes with overlapping clinical manifestations recently were shown by Turner et al. and Pettigrew et al. to be linked to markers at Xq26.     

Lujan-Fryns syndrome (mental retardation, X-linked, marfanoid habitus)

Kikuchi-Fujimoto disease (KFD) is a benign and self-limited disorder, characterized by regional cervical lymphadenopathy with tenderness, usually accompanied with mild fever and night sweats. Less frequent symptoms include weight loss, nausea, vomiting, sore throat. Kikuchi-Fujimoto disease is an extremely rare disease known to have a worldwide distribution with higher prevalence among Japanese and other Asiatic individuals. The clinical, histopathological and immunohistochemical features appear to point to a viral etiology, a hypothesis that still has not been proven. KFD is generally diagnosed on the basis of an excisional biopsy of affected lymph nodes. Its recognition is crucial especially because this disease can be mistaken for systemic lupus erythematosus, malignant lymphoma or even, though rarely, for adenocarcinoma. Clinicians' and pathologists' awareness of this disorder may help prevent misdiagnsois and inappropriate treatment. The diagnosis of KFD merits active consideration in any nodal biopsy showing fragmentation, necrosis and karyorrhexis, especially in young individuals presenting with posterior cervical lymphadenopathy. Treatment is symptomatic (analgesics-antipyretics, non-steroidal anti-inflammatory drugs and, rarely, corticosteroids). Spontaneous recovery occurs in 1 to 4 months. Patients with Kikuchi-Fujimoto disease should be followed-up for several years to survey the possibility of the development of systemic lupus erythematosus.     

Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression

We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6(3/4) pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6(3/4) pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.     

A mutation in the rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males

Heterozygous mutations in the X-linked MECP2 gene cause Rett syndrome, a severe neurodevelopmental disorder of young females. Only one male presenting an MECP2 mutation has been reported; he survived only to age 1 year, suggesting that mutations in MECP2 are male lethal. Here we report a three-generation family in which two affected males showed severe mental retardation and progressive spasticity, previously mapped in Xq27.2-qter. Two obligate carrier females showed either normal or borderline intelligence, simulating an X-linked recessive trait. The two males and the two obligate carrier females presented a mutation in the MECP2 gene, demonstrating that, in males, MECP2 can be responsible for severe mental retardation associated with neurological disorders.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

QRI8, a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae.

We have cloned and sequenced the gene encoding a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae. Disruption of this gene shows that it is not essential for cell viability.     

A gene for dominant nonspecific X-linked mental retardation is located in Xq28.

A large family (MRX48) with a nonspecific X-linked mental retardation condition is described. An X-linked semidominant inheritance is suggested by the segregation in three generations of a moderate to severe mental retardation in seven males and by a milder intellectual impairment in two females, without any specific clinical, radiological, or biological feature. Two-point linkage analysis demonstrated significant linkage between the disorder and several markers in Xq28 (maximum LOD score [Zmax] = 2.71 at recombination fraction [theta] = 0); multipoint linkage analyses confirmed the significant linkage with a Zmax of 3.3 at theta = 0, at DXS1684. A recombination event observed with the flanking marker DXS8011 delineates a locus between this marker and the telomere. The approximate length of this locus is 8-9 cM, corresponding to 5.5-6 Mb. In an attempt to explain the variable intellectual impairment in females, we examined X-chromosome inactivation in all females of the family. Inactivation patterns in lymphocytes were random or moderately skewed, and no correlation between the phenotypic status and a specific inactivation pattern was observed. The interval of assignment noted in this family overlaps with five MRX loci previously reported in Xq28.     

Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme)

The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.     

A new form of X-linked mental retardation with growth retardation, deafness, and microgenitalism.

The proband and two maternal uncles were similarly affected by a unique constellation of mental retardation and physical abnormalities. There were severe retardation, growth less than the third percentile, and significantly delayed bone age. They manifested deafness, a flat nasal bridge, several ocular abnormalities, and a rudimentary scrotum with cryptorchidism, and one had a small penis. The proband also had onychodystrophy of his fingers and toes. Their birth weights and lengths were less than expected. No chromosomal or biochemical abnormality was detected. Both uncles died, but the proband is healthy at 4 years. Their phenotype is distinguished from other forms of X-linked mental retardation and appears to be a new syndrome.     

Dissociation between mental retardation and fragile site expression in a family with fragile X-linked mental retardation

Summary We report an extended family in which two brothers with a fragile X chromosome are mentally retarded while a third brother with the fragile site is both phenotypically and mentally normal. The study of six probes detecting restriction fragment length polymorphisms on either sides of the fragile site Xq27 confirmed that the fragile X regions inherited by these three brothers were identical from DXS 102 to the telomere. These data highlight the heterogeneity of the fragile X syndrome, which is discussed in the framework of the different hypotheses previously proposed.     

A member of a gene family on Xp22.3, VCX-A, is deleted in patients with X-linked nonspecific mental retardation

X-linked nonspecific mental retardation (MRX) has a frequency of 0.15% in the male population and is caused by defects in several different genes on the human X chromosome. Genotype-phenotype correlations in male patients with a partial nullisomy of the X chromosome have suggested that at least one locus involved in MRX is on Xp22.3. Previous deletion mapping has shown that this gene resides between markers DXS1060 and DXS1139, a region encompassing approximately 1.5 Mb of DNA. Analyzing the DNA of 15 males with Xp deletions, we were able to narrow this MRX critical interval to approximately 15 kb of DNA. Only one gene, VCX-A (variably charged, X chromosome mRNA on CRI-S232A), was shown to reside in this interval. Because of a variable number of tandem 30-bp repeats in the VCX-A gene, the size of the predicted protein is 186-226 amino acids. VCX-A belongs to a gene family containing at least four nearly identical paralogues on Xp22.3 (VCX-A, -B, -B1, and -C) and two on Yq11.2 (VCY-D, VCY-E), suggesting that the X and Y copies were created by duplication events. We have found that VCX-A is retained in all patients with normal intelligence and is deleted in all patients with mental retardation. There is no correlation between the presence or absence of VCX-B1, -B, and VCX-C and mental status in our patients. These results suggest that VCX-A is sufficient to maintain normal mental development.