Molecular basis of Refsum disease: Sequence variations in Phytanoyl‐CoA Hydroxylase (PHYH) and the PTS2 receptor (PEX7)Jansen, Gerbert A.; Waterham, Hans R.; Wanders, Ronald J. A.
doi: 10.1002/humu.10315pmid: 14974078
Refsum disease has long been known to be an inherited disorder of lipid metabolism characterized by the accumulation of phytanic acid (3,7,11,15‐tetramethylhexadecanoic acid) caused by an α‐oxidation deficiency of this branched chain fatty acid in peroxisomes. The mechanism of phytanic acid α‐oxidation and the enzymes involved had long remained mysterious, but they have been resolved in recent years. This has led to the resolution of the molecular basis of Refsum disease. Interestingly, Refsum disease is genetically heterogeneous; two genes, PHYH (also named PAHX) and PEX7, have been identified to cause Refsum disease, as reviewed in this work. Hum Mutat 23:209‐218, 2004. © 2004 Wiley‐Liss, Inc.
Gross rearrangement breakpoint database (GRaBD™)Abeysinghe, Shaun S.; Stenson, Peter D.; Krawczak, Michael; Cooper, David N.
doi: 10.1002/humu.20006pmid: 14974079
Translocations and gross gene deletions are an important cause of both cancer and inherited disease. Such DNA rearrangements are nonrandomly distributed in the human genome as a consequence of selection for growth advantage and/or the inherent potential of some DNA sequences to be particularly susceptible to breakage and recombination. The Gross Rearrangement Breakpoint Database (GRaBD™; http://www.uwcm.ac.uk/uwcm/mg/grabd/) was established primarily for the analysis of the sequence context of translocation and deletion breakpoints in a search for characteristics that might have rendered these sequences prone to rearrangement. GRaBD, which contains 397 germline and somatic DNA breakpoint junction sequences derived from 219 different rearrangements underlying human inherited disease and cancer, is the only comprehensive collection of gross gene rearrangement breakpoint junctions currently available. Hum Mutat 23:219–221, 2004. © 2004 Wiley‐Liss, Inc.
Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapyKarageorgos, L.; Harmatz, P.; Simon, J.; Pollard, A.; Clements, P. R.; Brooks, D. A.; Hopwood, John J.
doi: 10.1002/humu.10313pmid: 14974081
Mucopolysaccharidosis type VI (MPS VI), or Maroteaux‐Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N‐acetylgalactosamine‐4‐sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356underscore;358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient. Hum Mutat 23:229–233, 2004. © 2004 Wiley‐Liss, Inc.
Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndromeLaccone, Franco; Jünemann, Ivonne; Whatley, Sharon; Morgan, Rhian; Butler, Rachel; Huppke, Peter; Ravine, David
doi: 10.1002/humu.20004pmid: 14974082
MECP2 mutations are responsible for Rett syndrome (RTT). Approximately a quarter of classic RTT cases, however, do not have an identifiable mutation of the MECP2 gene. We hypothesized that larger deletions arising from a deletion prone region (DPR) occur commonly and are not being routinely detected by the current PCR‐mediated screening strategies. We developed and applied a quantitative PCR strategy (qPCR) to samples referred for diagnostic assessment from 140 patients among whom RTT was strongly suspected and from a second selected group of 31 girls with classical RTT. Earlier MECP2 mutation screening in both groups of patients had yielded a wild‐type result. We identified 10 large deletions (7.1%) within the first group and five deletions in the second group (16.1%). Sequencing of the breakpoints in 11 cases revealed that eight cases had one breakpoint within the DPR. Among seven cases, the breakpoint distant to the DPR involved one of several Alu repeats. Sequence analysis of the junction sequences revealed that eight cases had complex rearrangements. Examination of the MECP2 genomic sequence reveals that it is highly enriched for repeat elements, with the content of Alu repeats rising to 27.8% in intron 2, in which there was an abundance of breakpoints among our patients. Furthermore, a perfect χ sequence, known to be recombinogenic in E. coli, is located in the DPR. We propose that the χ sequence and Alu repeats are potent factors contributing to genomic rearrangement. We suggest that routine mutation screening in MECP2 should include quantitative analysis of the genomic sequences flanking the DPR. Hum Mutat 23:234–244, 2004. © 2004 Wiley‐Liss, Inc.
A nicotine C‐oxidase gene (CYP2A6) polymorphism important for promoter activityPitarque, Marià; von Richter, Oliver; Rodríguez‐Antona, Cristina; Wang, Jue; Oscarson, Mikael; Ingelman‐Sundberg, Magnus
doi: 10.1002/humu.20002pmid: 14974084
In humans, several polymorphic variants have been described for the gene encoding the major nicotine C‐oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5′ flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5′‐upstream region of the CYP2A6 gene from eight individuals revealed a c.‐1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3′‐UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele‐specific hybridization (DASH) showed that, in both groups, the variants carrying the c.‐1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5′‐regulatory sequence showed that the region between c.‐1005 and c.‐1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.‐1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.‐1013A as compared to the c.‐1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.‐1005 and c.‐1019 in the CYP2A6 gene and that a c.‐1013A>G polymorphism in this region affects CYP2A6 transcription. Hum Mutat 23:258–266, 2004. © 2004 Wiley‐Liss, Inc.
Noonan syndrome–associated SHP2/PTPN11 mutants cause EGF‐dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activationFragale, Alessandra; Tartaglia, Marco; Wu, Jie; Gelb, Bruce D.
doi: 10.1002/humu.20005pmid: 14974085
Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain‐containing tyrosine phosphatase 2 (SHP2 or SHP‐2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome–causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal‐regulated kinase 2 activation, which was ligand‐dependent. Binding of SHP2 mutants to Grb2‐associated binder‐1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2‐associated binder‐1‐FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor‐mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal‐regulated kinase 2 activation. Taken together, these results document that Noonan syndrome‐associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N‐terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen‐activated protein kinase (ERK2/MAPK1) pathway in a ligand‐dependent manner that required docking through Grb2‐associated binder‐1 (GAB1), leading to increased cell proliferation. Hum Mutat 23:267–277, 2004. © 2004 Wiley‐Liss, Inc.
Analysis of CBP (CREBBP) gene deletions in Rubinstein‐Taybi syndrome patients using real‐time quantitative PCRCoupry, Isabelle; Monnet, Laurence; Moneim Attia, Azza Abd El; Taine, Laurence; Lacombe, Didier; Arveiler, Benoît
doi: 10.1002/humu.20001pmid: 14974086
Rubinstein‐Taybi syndrome (RTS) is a well‐defined syndrome characterized by facial abnormalities, broad thumbs, broad big toes, and growth and mental retardation as the main clinical features. RTS was shown to be associated with disruption of the CREB–binding protein gene CBP (CREBBP), either by gross chromosomal rearrangements or by point mutations. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (about 10%). Most deletion studies in RTS are performed by FISH analysis, and five cosmids must be used to cover the whole of the CBP gene, which spreads over 150 kb. Here we report the design of gene dosage assays by real‐time quantitative PCR that are targeted on three exons located respectively at the 5′ end (exon 2), in the middle (exon 12), and at the 3′ end (exon 30) of the CBP gene. This technique proved to be efficient and powerful in finding deletions and complementary to the other available techniques, since it allowed us to identify deletions at the 3′ end of the gene that had been missed by FISH analysis, and to refine some deletion breakpoints. Our results therefore suggest that real‐time quantitative PCR is a useful technique to be included in the deletion search in RTS patients. Hum Mutat 23:278–284, 2004. © 2004 Wiley‐Liss, Inc.