Rapid immunoblot and kinase assay tests for a syndromal form of X linked mental retardation: Coffin-Lowry syndrome

Coffin-Lowry syndrome (CLS) is a syndromal form of X linked mental retardation, in which some associated facial, hand, and skeletal abnormalities are diagnostic features. Accurate diagnosis, critical for genetic counselling, is often difficult, especially in early childhood. We have recently shown that Coffin-Lowry syndrome is caused by mutations in the gene encoding RSK2, a growth factor regulated protein kinase. RSK2 mutations are very heterogeneous and most of them lead to premature termination of translation or to loss of phosphotransferase activity or both. In the present study, we have evaluated immunoblot and RSK2 kinase assays as a rapid and simple diagnostic test for CLS, using cultured lymphoblastoid or fibroblast cell lines. Western blot analysis failed to detect RSK2 in six patients, suggesting the presence of truncated proteins in these patients. This conclusion was confirmed in four patients, in whom the causative mutations, all leading to premature termination of translation, were identified. Of four patients showing a normal amount of RSK2 protein on western blot and tested for RSK2 phosphotransferase activity, one had a dramatically impaired activity. Analysis of the RSK2 cDNA sequence in this patient showed a mutation of a putative phosphorylation site that would be critical for RSK2 activity. Preliminary results show that, at least, the western blot protocol can be successfully applied to lymphocyte protein extracts prepared directly from blood samples. These assays promise to become important diagnostic tools for CLS, particularly with regard to very young patients with no family history of the condition.     

Statistics of malignant neoplasms in children in Russia

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In a population of 172 healthy people (average age, 34; mutant frequency, 10.3 x 10(-6)), deletion/insertion mutations constituted 41% (89) of the 217 independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among +/- 1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3-200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by -1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both endpoints were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), possibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 deletions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation.     

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.     

Efficacy of gene testing for von Hippel-Lindau disease

OBJECTIVE: To determine the efficacy of genetic testing of individuals presenting with features possibly indicative of von Hippel-Lindau (VHL) disease, regardless of other relevant family and clinical details. SETTING AND PARTICIPANTS: Between September 1994 and December 1997, 16 unrelated individuals were referred to Genetic Services of Western Australia by local clinicians and by similar genetic services in other States, for VHL gene mutation analysis because of clinical manifestations suggestive of the diagnosis. METHODS: The subjects were investigated by screening for mutations in the polymerase chain reaction products of the three VHL gene exons using single-stranded conformational polymorphism analysis (SSCP). If no mutations were detected the exons were sequenced, and if no variations were found DNA was examined by Southern analysis for germinal rearrangements. RESULTS: Mutations in the VHL gene were detected in eight of 16 individuals (50%), including 3 individuals with no family history suggestive of VHL disease. Five mutations were detected by SSCP, two by gene sequencing and one by Southern analysis. Each mutation occurred only in a single family and three had not been previously reported. CONCLUSION: Genetic screening of individuals presenting with clinical features suggestive of VHL facilitates confirmation of the diagnosis, accurate genetic counselling and surveillance of at-risk family members. The necessity for costly and time-consuming screening programs can be reduced and screening directed at those carrying the mutation. Our low stringency criteria are justified for screening for VHL mutations.     

Diminished regional cerebral blood flow response to vibration in patients with blepharospasm

Mutations of the human Patched gene ( PTCH ) have been identified in individuals with the nevoid basal cell carcinoma syndrome (NBCCS) as well as in sporadic basal cell carcinomas and medulloblastomas. We have isolated a homologue of this tumour suppressor gene and localized it to the short arm of chromosome 1 (1p32.1-32.3). Patched 2 ( PTCH2 ) comprises 22 coding exons and spans approximately 15 kb of genomic DNA. The gene encodes a 1203 amino acid putative transmembrane protein which is highly homologous to the PTCH product. We have characterized the genomic structure of PTCH2 and have used single-stranded conformational polymorphism analysis to search for mutations in PTCH2 in NBCCS patients, basal cell carcinomas and in medulloblastomas. To date, we have identified one truncating mutation in a medulloblastoma and a change in a splice donor site in a basal cell carcinoma, suggesting that the gene plays a role in the development of some tumours.     

Genomic organization of the human TIMP-1 gene. Investigation of a causative role in the pathogenesis of X-linked retinitis pigmentosa 2

PURPOSE: To evaluate the role of TIMP-1 in inherited retinal degeneration. METHODS: The genomic structure of the TIMP-1 gene was established and male patients with x-linked retinitis pigmentosa 2 from five families were screened for sequence alterations by direct sequencing in all exons, exon-intron boundaries, and the 5' upstream region of the gene. RESULTS: TIMP-1 appears to be expressed in the retina at low levels and consists of six exons spanning a genomic region of approximately 4.5 kb on Xp11.23. No disease-specific sequence alterations were identified. A site substitution in exon 5 was observed in samples from control subjects and patients, but it did not alter the amino acid sequence of the protein product. CONCLUSIONS: The results of this study exclude mutations in the TIMP-1 coding sequence, splice sites, and the 5' upstream region as a cause of retinal degeneration in x-linked retinitis pigmentosa 2. However, an as yet unidentified regulatory element that lies outside these intervals may be implicated. The role of this tightly regulated protein in the normal functioning of the retina has yet to be determined.     

Dihydropyrimidinase deficiency: structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene

Dihydropyrimidinase (DHP) deficiency (MIM 222748) is characterized by dihydropyrimidinuria and is associated with a variable clinical phenotype. This disease might be associated with a risk of 5-fluorouracil toxicity, although no cases have been reported. We present here both the molecular characterization of the human DHP gene and, for the first time, the mutations causing DHP deficiency. The human DHP gene spans >80 kb and consists of 10 exons. It has been assigned to 8q22, by FISH. We performed mutation analysis of genomic DNA in one symptomatic and five asymptomatic individuals presenting with dihydropyrimidinuria. We identified one frameshift mutation and five missense mutations. Two related Japanese adult subjects were homozygous for the Q334R substitution, whereas two other, unrelated Japanese infant subjects were heterozygous for the same mutation, but this mutation is not common in the Japanese population. A Caucasian pediatric patient exhibiting epileptic attacks, dysmorphic features, and severe developmental delay was homozygous for W360R. Using a eukaryotic expression system, we showed that all mutations reduced enzyme activity significantly, indicating that these are crucial DHP deficiency-causing mutations. There was no significant difference, in residual activity, between mutations observed in the symptomatic and those observed in the asymptomatic individuals.     

Further delineation of renal-coloboma syndrome in patients with extreme variability of phenotype and identical PAX2 mutations

Renal-coloboma syndrome is a recently described autosomal dominant syndrome of abnormal optic nerve and renal development. Two families have been reported with renal-coloboma syndrome and mutations of the PAX2 gene. The PAX2 gene, which encodes a DNA-binding protein, is expressed in the developing ear, CNS, eye, and urogenital tract. Ocular and/or renal abnormalities have been consistently noted in the five reports of patients with renal-coloboma syndrome, to date, but PAX2 expression patterns suggest that auditory and CNS abnormalities may be additional features of renal-coloboma syndrome. To determine whether additional clinical features are associated with PAX2 mutations, we have used PCR-SSCP to identify PAX2 gene mutations in patients. We report here four patients with mutations in exon 2, one of whom has severe ocular and renal disease, microcephaly, and retardation, and another who has ocular and renal disease with high-frequency hearing loss. Unexpectedly, extreme variability in clinical presentation was observed between a mother, her son, and an unrelated patient, all of whom had the same PAX2 mutation as previously described in two siblings with renal-coloboma syndrome. These results suggest that a sequence of seven Gs in PAX2 exon 2 may be particularly prone to mutation.     

Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumors

The Beckwith-Wiedemann syndrome (BWS) is marked by fetal organ overgrowth and conveys a predisposition to certain childhood tumors, including Wilms tumor (WT). The genetics of BWS have implicated a gene that maps to chromosome 11p15 and is paternally imprinted, and the gene encoding the cyclin-cdk inhibitor p57KIP2 has been a strong candidate. By complete sequencing of the coding exons and intron/exon junctions, we found a maternally transmitted coding mutation in the cdk-inhibitor domain of the KIP2 gene in one of five cases of BWS. The BWS mutation was an in-frame three-amino-acid deletion that significantly reduced but did not fully abrogate growth-suppressive activity in a transfection assay. In contrast, no somatic coding mutations in KIP2 were found in a set of 12 primary WTs enriched for cases that expressed KIP2 mRNA, including cases with and without 11p15.5 loss of heterozygosity. Two other 11p15.5 loci, the linked and oppositely imprinted H19 and IGF2 genes, have been previously implicated in WT pathogenesis, and several of the tumors with persistent KIP2 mRNA expression and absence of KIP2 coding mutations showed full inactivation of H19. These data suggest that KIP2 is a BWS gene but that it is not uniquely equivalent to the 11p15.5 "WT2" tumor-suppressor locus.     

Characterization of mutations in the myotubularin gene in twenty six patients with X-linked myotubular myopathy

A candidate gene, myotubularin, involved in the pathogenesis of X-linked myotubular myopathy (MTM1) was isolated recently. Mutations originally were identified in 12% of patients examined for 40% of the coding sequence, raising the possibility that additional genes could be responsible for a proportion of X-linked cases. We report here the identification of mutations in 26 of 41 independent male patients with muscle biopsy-proven MTM, by direct genomic sequencing of 92% of the known coding sequence of the myotubularin gene. Eighteen patients had point mutations, including one A/G transition found in four patients which alters a splice acceptor site in exon 12 and leads to a three amino acid insertion. Six patients had small deletions involving <6 bp, while two larger deletions encompassed two or six exons, respectively. No differences were noted among the types of mutations between familial and sporadic cases. However, all of the five patients with a mild phenotype had missense mutations. While 50% of the mutations were found in exons 4 and 12, and three distinct mutations were found in more than one patient, no single mutation accounted for more than 10% of the cases. The low frequency of large deletions and the varied mutations identified suggest that direct mutation screening for molecular diagnosis may require gene sequencing.     

The GTP cyclohydrolase I gene in Russian families with dopa-responsive dystonia

OBJECTIVE: To search for mutations in the GTP cyclohydrolase I (GCH-I) gene in a set of Russian families with dopa-responsive dystonia (DRD). DESIGN: Six large families with 54 affected family members and 2 patients with sporadic DRD were examined. Mutation screening was performed using single-strand conformation polymorphism analysis followed by direct sequencing of the presumably mutated exons, in patients whose results showed a normal pattern on single-strand conformation polymorphism analysis, the entire coding region of the GCH-I gene was sequenced. RESULTS: Three new heterozygote point mutations located within exons 1, 2, and 4 of the GCH-I gene were identified in 3 families with autosomal-dominant inheritance. All these mutations are predicted to cause amino acid changes in the highly conserved regions of the gene. In patients from 3 other families and in both patients with sporadic DRD, no alterations in the translated portion of the GCH-I gene were observed. CONCLUSIONS: Mutations in the coding region of the GCH-I gene account for a significant fraction (up to half) of the patients with a typical clinical picture of DRD. None of the mutations in the GCH-I gene described so far were detected more than once, which precludes the possibility of creating simple DNA testing procedures for routine clinical practice.     

Cis- and trans-splicing and RNA editing are required for the expression of nad2 in wheat mitochondria

In wheat mitochondria, the gene coding for subunit 2 of the NADH-ubiquinone oxidoreductase (nad2) is divided into five exons located in two distant genomic regions. The first two exons of the gene, a and b, lie 22 kb downstream of exons c, d, and e, on the same DNA strand. All introns of nad2 are group II introns. A trans-splicing event is required to join exons b and c. It involves base pairing of the two precursor RNAs in the stem of domain IV of the intron. A gene coding for tRNA(Tyr) is located upstream of exon c. In addition to splicing processes, mRNA editing is also required for the correct expression of nad2. The mature mRNA is edited at 36 positions, distributed over its five exons, resulting in 28 codon modifications. Editing increases protein hydrophobicity and conservation.     

Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries

We identified two mutations in the CYP19 gene responsible for aromatase deficiency in an 18-year-old 46,XX female with ambiguous external genitalia at birth, primary amenorrhea and sexual infantilism, and polycystic ovaries. The coding exons, namely exons II-X, of the CYP19 gene were amplified by PCR from genomic DNA and sequenced directly. Direct sequencing of the amplified DNA from the patient revealed two single-base changes, at bp 1303 (C-->T) and bp 1310 (G-->A) in exon X, which were newly found missense mutations and resulted in codon changes of R435C and C437Y, respectively. Subcloning followed by sequencing confirmed that the patient is a compound heterozygote. The results of restriction fragment length polymorphism analysis and direct sequencing of the amplified exon X DNA from the patient's mother indicate maternal inheritance of the R435C mutation. Transient expression experiments showed that the R435C mutant protein had approximately 1.1% of the activity of the wild type, whereas C437Y was totally inactive. Cysteine-437 is the conserved cysteine in the heme-binding region believed to serve as the fifth coordinating ligand of the heme iron. To our knowledge, this patient is the first adult to have described the cardinal features of a syndrome of aromatase deficiency. Recognition that such defects exist will lead to a better understanding of the role of this enzyme in human development and disease.     

Therapeutic monitoring of tacrolimus in pediatric and adult transplanted patients

The gene that encodes the human alpha2 subunit of the inhibitory glycine receptor (GLRA2) is located on the X chromosome (Xp22.2) in a candidate region for a number of neurological disorders. Recently, an exclusion mapping strategy identified this region to be concordant in familial Rett syndrome (RTT) patients. Based on its established expression pattern and known function, GLRA2 was selected as a candidate gene for Rett syndrome. Major gene rearrangements were excluded based on Southern analysis using the GLRA2 cDNA as probe. To identify more subtle mutations, we determined the genomic structure for GLRA2, which consists of nine exons and a putative alternatively spliced exon 3. The exon-intron boundaries were sequenced in order to design primer sets for polymerase chain reaction (PCR) amplification of all exons and their immediately flanking intronic regions. PCR products amplified from genomic DNA isolated from 40 RTT patients were subsequently characterized by heteroduplex analysis, and no mutations were detected. Characterization of the intron-exon structure of GLRA2 will facilitate future mutational analysis of this gene for other neurological disorders mapping to human Xp22.2.     

p53 gene alterations in special types of breast carcinoma: a molecular and immunohistochemical study in archival material

The p53 locus on the short arm of chromosome 17 at 17p13.1 was examined for small genomic deletions and mutations in 23 formalin-fixed, paraffin-embedded cases of special types of breast carcinoma (six medullary, seven apocrine, five differentiated tubular, and five papillary). p53 mutations in the evolutionarily conserved exons 5-9 were detected in 11 cases (four apocrine, two papillary, two medullary, and three differentiated tubular), using the novel non-radioactive PCR-based Hydrolink mutation detection enhancement (MDE) method, and confirmed by direct sequencing of the PCR products. Missense mutations causing amino acid substitutions were evenly distributed among exons. One case of apocrine carcinoma showed a polymorphism at codon 213 (CGA-->CGG). Twelve out of 23 cases were found to express a strong nuclear signal against CM-1 and DO-7, two anti-p53-specific antibodies. Small genomic deletions in the vicinity of the p53 locus were detected in 11 tumours (three papillary, three differentiated tubular, two medullary, and three apocrine carcinomas), using the multiplex PCR method. No statistical correlation was found between deletions at 17p13.1 and p53 mutations (P < 0.5). In addition, p53 mutations and immunoexpression correlated with the c-erbB-2 gene product, an oncogenic protein that has been implicated in cell cycle control (P < 0.001). Our findings suggest that genomic alterations of the p53 gene are quite common events associated with special types of breast carcinoma, particularly of the apocrine subtype, but the prognostic value is unlikely to be of clinical importance.     

A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome

Kallmann's syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located on the Xp22.3 region, explaining the X-linked form of the disease. We report molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS. PCR of the 14 exons of the KAL gene was performed on genomic DNA. PCR products of all exons were purified and sequenced. Genetic defects in the KAL gene were found in 7 patients. One exhibits a deletion from exon 3 to exon 5. Six individuals present a previously unidentified missense mutation in exon 11, consisting of a G to A substitution at codon 514 (GAA to AAA). In the remaining 5 individuals, no mutations were observed. We also found three different polymorphic changes. The first one, in exon 2, had not been reported previously. The other two were located at exons 11 and 12. The deletion described, comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region that corresponds to the whey acidic protein motif. The same missense mutation was found in 6 of the 12 patients, indicating the possibility that it derived from a common ancestor or suggesting the presence of a hot spot in this region of the gene.     

High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing

Approximately 85% of patients with Alport syndrome (hereditary nephritis) have been estimated to have mutations in the X chromosomal COL4A5 collagen gene; the remaining cases are autosomal with mutations in the COL4A3 or COL4A4 genes located on chromosome 2. In the present work, the promoter sequence and previously unknown intron sequences flanking exons 2 and 37 of COL4A5 were determined. Furthermore, intron sequences flanking the other 49 exons were expanded from 35 to 190 to facilitate mutation analysis of the gene. Using this information, all 51 exons and the promoter region were PCR-amplified and sequenced from DNA of 50 randomly chosen patients with suspected Alport syndrome. Mutations were found in 41 patients, giving a mutation detection rate of 82%. Retrospective analysis of clinical data revealed that two of the cases might be autosomal. Although it could not be determined whether the remaining seven cases (14%) were autosomal or X chromosome-linked, it is likely that some of them were autosomal. It is concluded that PCR amplification and direct DNA sequencing of the promoter and exons is currently the best procedure to detect mutations in COL4A5 in Alport syndrome.