Amplification Of Pseudogenes Research Articles

New record of Carnidae (Diptera) from Taiwan and potential challenges in DNA barcode amplification due to pseudogene

The genus Carnus Nitzsch, 1818 comprises small ectoparasites that feed on the blood of juvenile avians. They are characterised by dealated adults with setose abdominal intersegmental membranes. Carnus orientalis Maa, 1968 was previously recorded in Malaysia and the Ryukyu Islands of Japan, parasitising two owl species: Ketupa ketupu (Horsfield, 1821) and Otus elegans (Cassin, 1852). This study confirms the occurrence of C. orientalis in Taiwan and presents a new host record, along with COI barcode sequences. Additionally, the study also elucidates the difficulties posed by blood meal contamination and pseudogene amplification as confounding factors intrinsic to the molecular taxonomic delineation of C. orientalis via universal DNA barcoding primers. The following new information regarding C. orientalis is provided in this study: Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan. Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling. Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon. New specific primers for the COI gene of Carnus were designed in this study. Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan. Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling. Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon. New specific primers for the COI gene of Carnus were designed in this study. The new distribution and ecological data of C. orientalis enhance our understanding of this species. The provision of new COI primers is anticipated to contribute to future studies employing DNA barcoding in bird-parasitic flies. The following new information regarding C. orientalis is provided in this study: Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan. Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling. Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon. New specific primers for the COI gene of Carnus were designed in this study. Carnus orientalis is first recorded in Taiwan, filling the gap in its East Asian distribution. This is also the first record of Carnidae from Taiwan. Otus lettia (Hodgson, 1836) (Aves, Strigidae) is reported as a new host for C. orientalis, identified on a fallen fledgling. Co-amplification of the host's COI is reported in this study using the universal PCR primer set LCO1490/HCO2198. Additionally, the amplification of a COI-like pseudogene using a newly-designed primer set is detected through abnormal translated amino acid sequences and the occurrence of a stop codon. New specific primers for the COI gene of Carnus were designed in this study. The new distribution and ecological data of C. orientalis enhance our understanding of this species. The provision of new COI primers is anticipated to contribute to future studies employing DNA barcoding in bird-parasitic flies.

Genotyping R1336X and Eliminating the Pseudogene Amplification in Type 3 von Willebrand Disease Patients

Genotyping R1336X and Eliminating the Pseudogene Amplification in Type 3 von Willebrand Disease Patients

Implication of Pseudo Reference Genes in Normalization of Data from Reverse Transcription-Quantitative PCR.

Pseudogenes are duplicated or retrotransposed DNA sequences of native functional genes. Amplification of pseudogenes along with gene of interest often produces false positive results, which is an innate problem in Reverse Transcription-quantitative PCR (RT-qPCR). Selecting a reference gene without any interference from pseudogene amplification is therefore a challenge to overcome. Among the common reference genes used for normalization (ACTB, GAPDH, HPRT1, TUBB, RNA18SN1 and B2M), B2M was found to have no pseudogenes in silico, which has also been confirmed by PCR and RT-qPCR. We also assessed the effect of pseudogenes on the determination of the stability of reference genes through data mining. The phylogenetic analysis of pseudogenes and functional genes revealed high sequence similarity among mammals. In addition, we demonstrated the deduction of pseudogene amplification signal using ValidPrime Assay (VPA) under conditions where genomic DNA contamination could not be avoided. Hence, we recommend the use of pseudo-free reference gene with consistent expression in the samples of interest or use VPA normalization method, where genomic DNA or pseudogene amplification is inevitable.

Pitfalls in molecular diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a putative error of metabolism with autosomal recessive heredity pattern. The main manifestations of classic form of CAH are salt-wasting, dehydration and simple virilization in both sexes and ambiguous genitalia in female gender. 21-hyroxylase (CYP21A2) impairment with prevalence value of 1 in 10,000–15,000 live births is the most common etiology of CAH. Because of consanguineous marriages, the frequency of the CAH in Iran is very high. A wide range of mutations diversity exists in CYP21A2 gene and a large number of these mutations derived from a highly homologous pseudogene, CYP21A1P, through gene conversion. In addition, new mutations such as small and large deletion and point mutations can also result in enzyme deficiency. Various methods for mutation detection were performed. The main obstacle in molecular diagnosis of CAH is amplification of pseudogene during polymerase chain reaction of CYP21A2. All attempts focus on discrimination of pseudogene from gene; that is why, there is the majority of mutations on pseudogene, and if we have contamination with the pseudogene, the result will be unreliable. Here, we discuss this methods and advantage and disadvantage of those.

Identification of novel PKD1 and PKD2 mutations in Korean patients with autosomal dominant polycystic kidney disease.

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder. It is caused by mutations in the PKD1 and PKD2 genes, and manifests as progressive cyst growth and renal enlargement, resulting in renal failure. Although there have been a few studies on the frequency and spectrum of mutations in PKD1 and PKD2 in Korean patients with ADPKD, only exons 36–46, excluding the duplicated region, were analyzed, which makes it difficult to determine accurate mutation frequencies and mutation spectra.MethodsWe performed sequence analysis of 20 consecutive unrelated ADPKD patients using long-range polymerase chain reaction (PCR) to avoid pseudogene amplification, followed by exon-specific PCR and sequencing of the all exons of these two genes. Multiplex ligation-dependent probe amplification was performed in patients in whom pathogenic mutations in PKD1 or PKD2 were not identified by LR-PCR and direct sequencing to detect large genomic rearrangements.ResultsAll patients met the diagnostic criteria of ADPKD, and pathogenic mutations were found in 18 patients (90.0%), comprising 15 mutations in PKD1 and three in PKD2. Among 10 novel mutations, eight mutations were found in the PKD1 gene while two mutations were found in the PKD2 gene. Eight of 14 PKD1 mutations (57.1%) were located in the duplicated region.ConclusionsThis study expands the spectra of mutations in the PKD1 and PKD2 genes and shows that the mutation frequencies of these genes in Korean ADPKD patients are similar to those reported in other ethnicities. Sequence analysis, including analysis of the duplicated region, is essential for molecular diagnosis of ADPKD.

Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome

To investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome. Invitro experiments. Academic medical center. One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome. Peripheral blood leukocytes were used to obtain DNA. Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities. We identified four KAL1 mutations (p.Met1?, p.Ala33Glyfs, p.Arg257*, and p.Trp462*) and two multiple exon deletions (exons 1-2 and 3-14) in six new male patients. Overall, 17 KAL1 defects (14.8%) were identified in the entire cohort of patients with Kallmann syndrome, including previously studied cases. KAL1-mutated patients presented with a more severe reproductive and nonreproductive phenotype (synkinesia, renal malformations, cryptorchidism, and anatomic olfactory abnormalities) in comparison with patients without KAL1 mutations. Intragenic deletions were one of the most often encountered defects (29.4%). Thesedeletions can be missed by polymerase chain reaction (PCR) due to Yq11.2 KAL1 pseudogene (KALP) spurious amplification. These results indicate that intragenic multiexon deletions are one of the most frequent KAL1 abnormalities, which can be more accurately detected by multiplex ligation-dependent probe amplification. In addition, KAL1 sequencing results should be interpreted with caution, and stringency conditions of the PCR reaction should be adjusted to avoid pseudogene amplification.

High-Resolution Melting Analysis as a Sensitive Prescreening Diagnostic Tool to Detect KRAS, BRAF, PIK3CA, and AKT1 Mutations in Formalin-Fixed, Paraffin-Embedded Tissues

As the availability of targeted therapies for several tumor types increases, the need for rapid and sensitive mutation screening is growing. KRAS mutations constitutively activate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway and therefore play an important role in anti-epidermal growth factor receptor therapy for patients with colorectal cancers. Mutationally activated PIK3CA and AKT1 genes are promising therapeutic targets in breast cancer. In 60% to 70% of malignant melanomas, a mutation in BRAF can be found. Thus, the blocking of the oncogenic signaling induced by this mutation is now used as treatment approach. To establish high-resolution melting assays for routinely used predictive analyses of KRAS , AKT1 , PIK3CA , and BRAF mutations. High-resolution melting assays were developed by using specifically designed primers and genomic DNA isolated either from cell lines or formalin-fixed paraffin-embedded tissues, oligonucleotides, or plasmids. Melting curve analyses were performed on the LightCyler platform and mutation analyses were additionally confirmed by Sanger sequencing. We developed high-resolution melting assays by using genomic DNA containing the desired mutation, which enabled us to detect percentages of mutated DNA (3.1% to 12.5%) mixed in a wild-type background. Assays were evaluated by hybridization probes and/or Sanger sequencing to exclude pseudogene amplification. The high-resolution melting assays were validated with genomic DNA from different tumor entities. The concordance between Sanger sequencing and high-resolution melting was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for the remaining assays. High-resolution melting provides a valid and powerful tool for detecting genomic mutations efficiently.

Abstract 2108: Prevalence of mutations in MSH6, PMS1 and PMS2 gene in Brazilian families with Lynch syndrome

Abstract Background: Lynch syndrome (LS) is an autosomal dominant disorder predisposing to colorectal cancer and increased risk for cancers of the stomach, small intestine, hepatobiliary system, kidney, ureter, ovary, and sebaceous tumors. Germline mutations in the MMR genes MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), post- meiotic segregation increased 1 (PMS1) or post- meiotic segregation increased 2 (PMS2), are known to cause LS. While mutations in MLH1 and MSH2 gene, account for 70% - 80% of all Lynch syndrome colorectal cancers, the involvement of the MSH6, PMS2 and PMS1 gene is continually rising. Altogether, MSH6, PMS1 and PMS2 mutations account for 5-20% of kindreds in which MSH2 and MLH1 mutations are excluded. Objective: The aim of the study was evaluate the frequency of mutations in MSH6, PMS1 and PMS2 gene. Methods: Seventy one unrelated patients without MLH1/MSH2 mutations were eligible for this study. Genomic DNA was obtained from peripheral blood and primers were designed within introns of all exons of MSH6, PMS1 gene. In order to avoid the amplification of pseudogenes for the PMS2 gene, a set of four primer pairs were design. Results: Out of 71 patients, 20 fulfilled the Amsterdam criteria and other 51 fulfilled the Bethesda guideline. Four novel pathogenic mutations were detected, three in MSH6 and 1 in PMS2. In addition, 7 variants of uncertain significance were encountered, in which two of them were previously described as pathogenic. No mutations were found in PMS1 gene. Conclusion: Our data shows that 8.5% (6/71) of the individuals have mutations in MSH6 and PMS2 gene and these results strongly suggest that MSH6 and PMS2 should not be excluded from the MMR screening in Brazilian families suspected for Lynch syndrome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2108. doi:1538-7445.AM2012-2108

Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations

This study describes how the new massive parallel sequencing technology can be implemented in a diagnostic setting for the breast cancer susceptibility genes (BRCA1 and BRCA2). The throughput was maximized by increasing uniformity in coverage, obtained by a multiplex approach, which outperformed pooling of singleplex PCRs. We evaluated the sensitivity by analysis of 133 distinct sequence variants; three (2%) deletions or duplications in homopolymers of greater than or equal to seven nucleotides remained undetected, illustrating a limitation of pyrosequencing. Furthermore, other limitations like nonrandom sequencing errors, pseudogene amplification, and failure to detect multiexon deletions are thoroughly described. Our workflow illustrates the potential of massive parallel sequencing of large genes in a diagnostic setting, which is of great importance to meet the increasing expectations of genetic testing. Implementation of this approach will hopefully lead to a strong reduction in turnaround times. As a consequence a wider spectrum of at risk women will be able to benefit from therapeutic interventions and prophylactic interventions.

Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

A cross-sectional analysis of stored Ziehl-Neelsen (ZN)-stained sputum smear slides (SSS) obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC). A two-step approach was used to distinguish M. bovis from other members of MTC: (i) oxyR pseudogene amplification to detect MTC and, subsequently, (ii) allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5%) SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB) results and the source laboratory were associated (p < 0.05) with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.

Towards the simplification of MHC typing protocols: targeting classical MHC class II genes in a passerine, the pied flycatcher Ficedula hypoleuca

BackgroundMajor Histocompatibility Complex (MHC) has drawn the attention of evolutionary biologists due to its importance in crucial biological processes, such as sexual selection and immune response in jawed vertebrates. However, the characterization of classical MHC genes subjected to the effects of natural selection still remains elusive in many vertebrate groups. Here, we have tested the suitability of flanking intron sequences to guide the selective exploration of classical MHC genes driving the co-evolutionary dynamics between pathogens and their passerine (Aves, Order Passeriformes) hosts.FindingsIntronic sequences flanking the usually polymorphic exon 2 were isolated from different species using primers sitting on conserved coding regions of MHC class II genes (β chain). Taking the pied flycatcher Ficedula hypoleuca as an example, we demonstrate that careful primer design can evade non-classical MHC gene and pseudogene amplification. At least four polymorphic and expressed loci were co-replicated using a single pair of primers in five non-related individuals (N = 28 alleles). The cross-amplification and preliminary inspection of similar MHC fragments in eight unrelated songbird taxa suggests that similar approaches can also be applied to other species.ConclusionsIntron sequences flanking the usually polymorphic exon 2 may assist the specific investigation of classical MHC class II B genes in species characterized by extensive gene duplication and pseudogenization. Importantly, the evasion of non-classical MHC genes with a more specific function and non-functional pseudogenes may accelerate data collection and diminish lab costs. Comprehensive knowledge of gene structure, polymorphism and expression profiles may be useful not only for the selective examination of evolutionarily relevant genes but also to restrict chimera formation by minimizing the number of co-amplifying loci.

Clinical relevance of germ cell cancer cells detected by real time PCR in apheresis products of poor risk patients undergoing high dose chemotherapy.

4579 Background: The clinical relevance of germ cell tumor (GCT)-specific mRNA in peripheral blood stem cell (PBSC) harvests of germ-cell cancer patients undergoing high dose chemotherapy (HDCT) is controversial, possibly caused by inappropriate PCR conditions with inadequate sensitivity and specificity and small numbers of patients analyzed. We re-evaluated this issue in a large number of PBSC-products using a novel, GCT-specific quantitative PCR assay. Methods: PBSC-harvests of 140 patients treated with HDCT for relapsed or platinum-refractory GCT were tested for the presence contaminating tumor cells using a highly tumor cell-specific PCR protocol, which amplified the un-conserved 5’-untranslated region (5’UTR) of the germ cell tumor-specific alkaline phosphatase (gcap) gene, but not alkaline phosphatase mRNA, the latter being also present in the peripheral blood of healthy donors. In order to further avoid false positive results by amplification of pseudogenes, all mRNA's were subjected to repeated DNAse digestion before entering cDNA synthesis. Results: The novel 5’UTR gcap specific PCR enabled the quantitative detection of gcap mRNA with an unprecedented specificity and good sensitivity of 10^-5. A total of 140 PBSC apheresis products were screened, with sufficient mRNA and clinical data available from 113 patients. The median time to progression (TTP) was significantly shorter in patients with gcap-positive (n=66) as compared to gcap-negative PBSC products (n=44) (7.0 months versus not reached, p=0.01). With a median follow up of 8 years, the median overall survival was shorter in patients with gcap-positive as compared to gcap-negative PBSC products (20 month vs. not reached). This difference did not reach statistical significance (p=0.08). Conventional qualitative gcap-PCR failed to separate cohorts with different prognosis, underscoring the importance of a validated quantitative PCR assay to address the clinical impact of PBSC tumor cell contamination in GCT. Conclusions: A quantitative and gcap-specific 5’UTR PCR assay detecting gcap-mRNA in PBSC-harvests is a strong adverse prognostic factor in GCT patients undergoing HDCT. No significant financial relationships to disclose.

Novel keratin 14 hotspot mutation in Dowling-Meara type of epidermolysis bullosa simplex: Strategy to avoid KRT14 pseudogene amplification by a simple approach

Novel keratin 14 hotspot mutation in Dowling-Meara type of epidermolysis bullosa simplex: Strategy to avoid KRT14 pseudogene amplification by a simple approach

Novel Mutations in Types 2 & 3 von Willebrand Disease and Correlation with von Willebrand Factor Multimer Patterns.

Background & Objectives: Diagnosis and classification of VWD are currently based on integration of personal and family bleeding histories and results of protein-based diagnostic tests (VWF:Ag, VWF:RCo, FVIII:C, VWF:CB and VWF multimers) which have performance limitations. Genetic testing is emerging as a complementary diagnostic tool. We have identified mutations and correlated VWF multimer patterns in our patients (pt) with types 2 & 3 VWD, and report novel candidate mutations.Patients & Methods: Unrelated pt with type 3 (n=6) and subtypes 2 (n=22) VWD, from Mayo Comprehensive Hemophilia Center, consented to this IRB approved study. PCR amplification, from genomic DNA, of VWF gene (splice junctions, coding and promoter regions), avoiding pseudogene amplification, followed by ABI® sequencing and analysis (Mutation Surveyor: SoftGenetic®) were performed. Mutations were compared to VWD (ISTH), NCBI NR nucleotide and DV SNP databases. Comparison with available VWF sequences from other vertebrate species was performed. Selected regions not known to contain mutations remain to be sequenced. VWF multimer analysis was performed using a novel in-gel immunostaining and infrared imaging system.Results: Type 2A VWD (n=10), a novel mutation in exon 28 (E28) (V1524G: 2 pt) with characteristic VWF multimer abnormalities (decreased HMWM, increased satellite banding, suggesting enhanced VWF proteolysis). Type 2B VWD (n=6), 2 pt were compound heterozygous for 2 previously reported mutations, the first in E28 (R1306W)/E48 (T2647M) and the second in E28 (V1316M)/E20 (R854Q). Type 2M VWD (n=4):a.an 84 yr female with personal (spontaneous and post-surgical) and family bleeding histories, VWF:RCo 72%, VWF:Ag 118% (RCo:Ag ratio 0.61), normal FVIII and platelets, and aberrant VWF multimer banding pattern without substantive reduction of HMWM, had a novel mutation in E52 (S2775C; conserved in mouse, rat, chicken and dog);b.a 14 yr female with a history of bleeding, VWF:RCo 34%, VWF:Ag 68% (RCo:Ag ratio 0.5) and normal FVIII and platelets, was compound heterozygous for a known mutation (R1399C subtype not classified in VWD database) and two novel mutations: E30 (P1725S) (conserved in mouse, rat, dog, cow and chimp) & E49 (T2666M) (conserved in mouse, dog, rat not in chicken). VWF multimers demonstrated abnormal banding pattern without substantive reduction of HMWM;c.2 unrelated pt and families with ultra-large and smeary multimers, previously classified as Vicenza variant, had R1374C mutations and were reclassified as Type 2M VWD. Type 2N VWD (n=1) previously reported mutation E20 (R854Q) was confirmed.Type 3 VWD, 2 pt had novel mutations, the first in E22 (E950X) and the second was a compound heterozygote for E43 (R2478Q)/E40 (E2322V) mutations. In general, patients with similar mutations had similar multimer patterns.Conclusions: We report novel candidate mutations in types 2 & 3 VWD and demonstrate novel VWF multimer patterns with our in-gel staining system. Multimer patterns correlated well with the underlying genotype. The novel mutations are likely causative, given their occurrence at highly conserved residues, but this needs to be confirmed with expression studies. Integration of genetic testing into the diagnostic workup of VWD will potentially lead to more accurate diagnosis and subtyping of VWD, and may further refine protein based testing and provide additional biological insight into VWD.

Porcine MHC Class II Haplotype Divergence (35.15)

Abstract The swine major histocompatibility complex (MHC) located in SSC7, is a gene dense region with a role in antigen presentation and controlling immune responsiveness. Although highly polymorphic, serological approaches to identify and define alleles have been disappointing. To determine the extent of MHC polymorphisms of class II genes, DNA from divergent world breeds of unrelated pigs was analyzed for SNPs. Primers targeting exon 2 of DQB and DRB were shown to be locus specific and amplified both loci for each animal. Expression of exon 2 PCR amplicons for both genes was verified through sequence analysis revealing 30 haplotypes comprised of over 20 different alleles among the 9 breeds. Located within the two genes were a total of 24 polymorphic sites, 22 of these were located within the antigen recognition site (DRB had two in the alpha helix) where 17 of those were nonsynonymous substitutions (7 synonymous). The breeds analyzed were selected to represent geographic areas in order to determine if haplotype divergence correlated with location of origin. The first group was identified as “Asian” and included Meishan, Erhualian and Lanyu breeds. The second group represented the “British Isles” (Hampshire, Yorkshire and Berkshire breeds) and the third group was identified as “Continental” (Duroc, Large White, and Landrace breeds). Fewer breed specific alleles were observed in breeds selected for lower fat and longer carcasses. SNPs in DRB appear to be under positive Darwinian selection with a dN/dS value of 13 as opposed to DQB with a value of 1 (no selective pressure or purifying selection) To confirm expression of the PCR products and eliminate the possibility of pseudogenes, RT-PCR with primers from intron-exon boundaries were utilized and verified the absence of pseudogene amplification and revealed highest RNA expression in DRB especially in heart, bone marrow, lung, small intestine and lymph node.

Comprehensive Mutation Scanning of NF1 in Apparently Sporadic Cases of Pheochromocytoma

Pheochromocytoma is a rare manifestation in patients with neurofibromatosis type 1 (NF 1). The 57-exon susceptibility gene NF1 has so far not been systematically scanned for unexpected germline mutations in individuals with sporadic pheochromocytoma. Twenty-seven patients with bilateral adrenal and/or extraadrenal abdominal pheochromocytoma not carrying germline mutations of the genes VHL, RET, SDHB, and SDHD were selected from the European-American pheochromocytoma registry. All 57 exons and flanking intronic regions of the NF1 gene were PCR amplified using newly designed primer pairs to exclude the amplification of pseudogenes. Intragenic mutation scanning was performed using denaturing HPLC and bidirectional direct sequencing. Of the 27 apparently sporadic cases, one (4%) was found to have a pathogenic germline NF1 mutation, Leu303Arg. Clinical reevaluation of this individual, who had bilateral pheochromocytoma, revealed classic, but very mild, features of NF 1, one cutaneous neurofibroma, axillary freckling, and Lisch nodules of the iris as well as a few café-au-lait spots. In the absence of germline mutations in VHL, RET, SDHD, and SDHB, patients with pheochromocytoma, especially with bilateral disease, should be checked thoroughly for clinical lesions suggestive of underlying syndromes such as the cutaneous and ophthalmological features characteristic of NF 1.

Mutation of the tumour suppressor p33 ING1 b is rare in melanoma

The p33ING1b gene is involved in the p53-dependent response to DNA damage following exposure to ultraviolet radiation, and has recently been reported to be mutated in 20% of melanoma tumours. We sought to assess the p33ING1b mutation rate in our large panels of fresh melanomas and melanoma cell lines. We screened 83 primary melanomas and 55 melanoma cell lines for mutations in p33ING1b by single-strand conformational polymorphism analysis and by direct sequencing. In contrast to previous reports, we found no somatic p33ING1b mutations in our panel of melanomas. We found that some of the discrepancy between our results and previously published studies may be due to inadvertent amplification of the ING1 pseudogene (INGX), and/or contamination of some samples with murine Ing1. p33ING1b mutations in melanoma are rare. We have highlighted the importance of allele-specific primer design to avoid pseudogene amplification, and also the necessity to confirm the genetic identity and species of origin of individual cell lines. Further studies are needed to clarify the possible role of p33ING1b in melanoma tumorigenesis.

Two pathogenic point mutations exist in the authentic mitochondrial genome, not in the nuclear pseudogene.

Technical advancements in molecular genetics have shown various mitochondrial DNA (mtDNA) abnormalities in patients with mitochondrial myopathies. Recently, it has been revealed that, in these patients, the nuclear DNA carries sequences similar to those of the mtDNA (nuclear pseudogene) and it has several point mutations previously reported to be pathogenic. We verified the existence of the T3250C and T3291C mutations, which we have found in patients with mitochondrial myopathy, in the authentic mitochondrial genome. A long polymerase chain reaction provides a powerful tool for avoiding nuclear pseudogene amplification and for ruling out ambiguity in the detection of the mutation for diagnosis.

Allele-Specific Amplification for the Diagnosis of Autosomal Recessive Spinal Muscular Atrophy

The SMN1 gene is homozygously deleted for at least exon 7, interrupted or converted to a non-functional telomeric copy in most cases of proximal spinal muscular atrophies. The presence of a pseudogene hampers direct detection of the exon 7 deletion. We describe a method for the detection of the of exon 7 deletion, based on the amplification refractory mutation system (ARMS), in a multiplex PCR with fluorescent-labelled primers. The gene and pseudogene amplification products differ in the dye bound and in their size, which allows distinction of both products on electrophoresis. The pseudogene is used as an internal control, and this method gives a clear and specific pattern for the patients. Amplification is achieved with 30 cycles, and specificity is retained up to 40 cycles.