Identification of UBA1 as the causative gene of an X-linked non-Kennedy spinal-bulbar muscular atrophy.

BACKGROUND AND PURPOSE: Spinal-bulbar muscular atrophy (SBMA) (Kennedy's disease) is a motor neuron disease. Kennedy's disease is nearly exclusively caused by mutations in the androgen receptor encoding gene (AR). The results of studies aimed at identification of the genetic cause of a disease that best approximates SBMA in a pedigree (four patients) without mutations in AR are reported. METHODS: Clinical investigations included thorough neurological and non-neurological examinations and testing. Genetic analysis was performed by exome sequencing using standard protocols. UBA1 mutations were modeled on the crystal structure of UBA1. RESULTS: The clinical features of the patients are described in detail. A missense mutation in UBA1 (c.T1499C; p.Ile500Thr) was identified as the probable cause of the non-Kennedy SBMA in the pedigree. Like AR, UBA1 is positioned on chromosome X. UBA1 is a highly conserved gene. It encodes ubiquitin-like modifier activating enzyme 1 (UBA1) which is the major E1 enzyme of the ubiquitin-proteasome system. Interestingly, UBA1 mutations can also cause infantile-onset X-linked spinal muscular atrophy (XL-SMA). The mutation identified here and the XL-SMA causative mutations were shown to affect amino acids positioned in the vicinity of UBA1's ATP binding site and to cause structural changes. CONCLUSION: UBA1 was identified as a novel SBMA causative gene. The gene affects protein homeostasis which is one of most important components of the pathology of neurodegeneration. The contribution of this same gene to the etiology of XL-SMA is discussed.

HOX cluster and their cofactors showed an altered expression pattern in eutopic and ectopic endometriosis tissues.

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology.

Confident gene activity prediction based on single histone modification H2BK5ac in human cell lines.

BACKGROUND: The histones in the core of nucleosomes may be subject to covalent post-transcriptional modifications. These modifications are thought to correlate with and possibly affect various genomic functions, including transcription. Each modification may alone or in combination with other modifications influence or be influenced by transcription. We aimed to identify correlations between single modifications or combinations of modifications at specific nucleosome sized gene regions with transcription activity based on global histone modification and transcription data of human CD4+ T cells and three other human cell lines. Transcription activity was defined in a binary fashion as either on or off. The analysis was done using the Classification and Regression Tree (CART) data mining protocol, and the Multifactorial Dimensionality Reduction (MDR) method was performed to confirm the CART results. These powerful methods have not previously been used for analysis of histone modification data. RESULTS: We showed that analysis of the single histone modification H2BK5ac at only four gene regions correctly predicted transcription activity status of over 75% of genes in CD4+ T-cells. The H2BK5ac modification status also had high power for prediction of gene transcription activity in the three other cell lines studied. The informative gene regions with the H2BK5ac modification were all positioned proximal to transcription initiation sites. The CART and MDR methods were appropriate tools for the analysis performed. In the study, we also developed a non-arbitrary protocol for binary classification of genes as transcriptionally active or inactive. CONCLUSIONS: The importance of H2BK5ac modification with regards to transcription control has not previously been emphasized. Analysis of this single modification at only four nucleosome sized gene regions, all of which are at or proximal to transcription initiation, has high power for prediction of gene transcription activity.

The novel mutation p.Asp251Asn in the ß-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine.

SUCLA2 is one of several nuclear-encoded genes that can cause encephalomyopathy accompanied by mitochondrial DNA depletion. The disorder usually manifests in early childhood and leads to early death. The gene encodes one of the subunits of succinyl-CoA synthase, the enzyme that catalyzes the reversible conversion of substrates succinyl-CoA and ADP to products succinate and ATP in the tricarboxylic acid pathway. Thirty-two individuals harboring mutations in SUCLA2 have so far been reported, and five different mutations were observed among these individuals. Here we report identification of a novel mutation in SUCLA2 in two cousins affected with encephalomyopathy. The novel mutation causes p.Asp251Asn; the affected amino acid is likely positioned within the ATP-grasp domain of the encoded protein. As previously reported in other patients, we did not observe elevation of methylmalonic acid, the biochemical hallmark of patients with mutations in SUCLA2. We instead found elevated levels of succinylcarnitine.

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6.

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue.

Insight into epigenetics of human endometriosis organoids: DNA methylation analysis of HOX genes and their cofactors.

OBJECTIVE: To evaluate and compare the methylation pattern of Human Homeobox (HOX) clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial tissues with ectopic and eutopic endometriosis organoids as advanced preclinical research models. DESIGN: A chromatin immunoprecipitation (ChIP) array containing 84 genes was used to analyze methylation levels of HOX clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial biopsy specimens as well as ectopic and eutopic endometriosis organoids. SETTING: Reproductive biomedicine and cell science research centers. PATIENT(S): Nine healthy women without endometriosis (control) and 16 women diagnosed with endometriosis. INTERVENTION(S): Ectopic endometrial lesions were obtained using a laparoscopic procedure, and eutopic and control endometrium biopsy specimens were obtained using pipelle sampling. MAIN OUTCOME MEASURE(S): Methylation levels of HOX clusters (A-D) and HOX cofactors in eutopic and ectopic endometrial biopsy specimens, as well as eutopic and ectopic endometriosis organoids and normal endometrium. RESULT(S): Most HOX clusters (A-D) and HOX cofactors showed methylation alterations in ectopic/eutopic endometrial tissues and ectopic/eutopic endometriosis organoids compared with normal endometrium. These methylation alterations had the same pattern in ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids in most genes. A contrariwise methylation pattern was observed in 28 of 84 genes in the ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids. CONCLUSION(S): Because a conserved pattern of methylation alterations in endometriosis tissues and organoids was observed for most of the investigated genes (56 of 84), it can be concluded that endometriosis organoids maintain epigenetic changes. Therefore, our study suggests endometriosis organoids as a novel preclinical model to determine the epigenetic mechanisms that underlie endometriosis.

Genotyping results of Iranian PCG families suggests one or more PCG locus other than GCL3A, GCL3B, and GCL3C exist.

PURPOSE: To assess whether loci other than GLC3A, GLC3B, and GLC3C are linked to primary congenital glaucoma (PCG). METHODS: The gene CYP1B1 at GLC3A was screened in 19 Iranian PCG probands who had been recruited mostly from among individuals of Turkish ethnicity and individuals from central and eastern Iran. The gene MYOC was screened in patients from this cohort who lacked CYP1B1 mutations and in ten patients previously shown not to carry CYP1B1 mutations. Family members of 19 probands without mutations in either of these genes were recruited for assessment of linkage to GLC3B and GLC3C by genotyping microsatellite markers. In total, 127 individuals, including 35 affected with PCG, were genotyped. RESULTS: Eleven (57.9%) of the newly recruited PCG patients did not carry disease-associated mutations in CYP1B1. Disease-associated MYOC mutations were not observed in any of the patients screened. Inheritance of PCG in all the families was consistent with an autosomal recessive pattern. Linkage to GLC3B and GLC3C was ruled out in nine of the families on the basis of autozygosity mapping and haplotype analysis. CONCLUSIONS: Observation of the absence of linkage to GLC3B and GLC3C in at least nine families without CYP1B1 mutations suggests that at least one PCG-causing locus other than GLC3A, GLC3B, and GLC3C may exist. The disease-causing gene or genes in the novel locus or loci may account for PCG in a notable fraction of Iranian patients.

Contributions of MYOC and CYP1B1 mutations to JOAG.

PURPOSE: To investigate the role of MYOC and CYP1B1 in Iranian juvenile open angle glaucoma (JOAG) patients. METHODS: Twenty-three JOAG probands, their available affected and unaffected family members, and 100 ethnically matched control individuals without history of ocular disease were recruited. Clinical examinations of the probands included slit lamp biomicroscopy, intraocular pressure (IOP) measurement, gonioscopic evaluation, fundus examination, and perimetry measurement. Familial cases were classified according to the mode of inheritance. Exons of MYOC and CYP1B1 were sequenced, and novel variations assessed in the control individuals. Potential disease-associated variations were tested for segregation with disease status in available family members. RESULTS: The mode of inheritance of the disease in the families of four probands (17.4%) appeared to be autosomal dominant and in at least eight (34.8%) to be autosomal recessive. Four patients carried MYOC mutations, and an equal number carried CYP1B1 mutations. The MYOC mutations were heterozygous; two of them (p.C8X and p.L334P) are novel, and one codes for the shortest truncated protein so far reported. Autosomal recessive inheritance was consistent with inheritance observed in families of patients carrying CYP1B1 mutations. All these patients carried homozygous mutations. CONCLUSIONS: MYOC and CYP1B1 contributed equally to the disease status of the Iranian JOAG patients studied. The contribution of the two genes appeared to be independent in that no patient carried mutations in both genes. The fraction of Iranian patients carrying MYOC mutations was comparable to previously reported populations.

CYP1B1 mutation profile of Iranian primary congenital glaucoma patients and associated haplotypes.

The mutation spectrum of CYP1B1 among 104 primary congenital glaucoma patients of the genetically heterogeneous Iranian population was investigated by sequencing. We also determined intragenic single nucleotide polymorphism (SNP) haplotypes associated with the mutations and compared these with haplotypes of other populations. Finally, the frequency distribution of the haplotypes was compared among primary congenital glaucoma patients with and without CYP1B1 mutations and normal controls. Genotype classification of six high-frequency SNPs was performed using the PHASE 2.0 software. CYP1B1 mutations in the Iranian patients were very heterogeneous. Nineteen nonconservative mutations associated with disease, and 10 variations not associated with disease were identified. Ten mutations and three variations not associated with disease were novel. The 13 novel variations make a notable contribution to the approximately 70 known variations in the gene. CYP1B1 mutations were identified in 70% of the patients. The four most common mutations were G61E, R368H, R390H, and R469W, which together constituted 76.2% of the CYP1B1 mutated alleles found. Six unique core SNP haplotypes were identified, four of which were common to the patients with and without CYP1B1 mutations and controls studied. Three SNP blocks determined the haplotypes. Comparison of haplotypes with those of other populations suggests a common origin for many of the mutations.

Sex Bias in Primary Congenital Glaucoma Patients with and without CYP1B1 Mutations.

PURPOSE: To investigate variations in sex ratio among Iranian primary congenital glaucoma (PCG) patients with and without mutations in the CYP1B1 gene and to evaluate possible clinical variations associated with sex in these two groups. METHODS: Phenotypical data on 104 unrelated Iranian PCG patients who had previously been screened for CYP1B1 mutations were analyzed. Emphasis was placed on analysis of sex ratios among patients with and without CYP1B1 mutations. In addition to sex, familial and sporadic incidence and clinical features including age at onset, bilateral/unilateral involvement, corneal diameter, intraocular pressure, and cup-disc ratios were compared between these two groups. Information on phenotypical parameters was available for most but not all patients. RESULTS: Among the 93 PCG patients whose sex was recorded, 57 were male (61.3%) and 36 were female (38.7%) (P=0.03). Patients with CYP1B1 mutations included 37 male (66.1%) and 29 female (43.9%) subjects (P=0.30), while patients without the mutation included 20 (74.1%) male and 7 (25.9%) female individuals (P=0.013). Our data did not provide conclusive evidence on difference in severity of the disease between those with and without CYP1B1 mutations, nor between the two sexes. CONCLUSION: Consistent with data on PCG patients from other populations, the overall incidence of PCG in Iran seems to be higher among male subjects. The difference in incidence between the two sexes was not significant among patients whose disease was due to mutations in CYP1B1. The overall higher incidence of PCG among male subjects seems to be attributable to a higher incidence in male patients not harboring CYP1B1 mutations, suggesting that other genes or factors may be involved in manifestation of PCG phenotypes in a sex dependent manner.