Mutation Screening In Patients Research Articles

Identification of a shared, common haplotype segregating with an SGCB c.544 T > G mutation in Indian patients affected with sarcoglycanopathy

Sarcoglycanopathy is the most frequent form of autosomal recessive limb-girdle muscular dystrophies caused by mutations in SGCB gene encoding beta-sarcoglycan proteins. In this study, we describe a shared, common haplotype co-segregating in 14 sarcoglycanopathy cases from 13 unrelated families from south Indian region with the likely pathogenic homozygous mutation c.544 T > G (p.Thr182Pro) in SGCB. Haplotype was reconstructed based on 10 polymorphic markers surrounding the c.544 T > G mutation in the cases and related family members as well as 150 unrelated controls from Indian populations using PLINK1.9. We identified haplotype H1 = G, A, G, T, G, G, A, C, T, G, T at a significantly higher frequency in cases compared to related controls and unrelated control Indian population. Upon segregation analysis within the family pedigrees, H1 is observed to co-segregate with c.544 T > G in a homozygous state in all the pedigrees of cases except one indicating a probable event of founder effect. Furthermore, Identical-by-descent and inbreeding coefficient analysis revealed relatedness among 33 new pairs of seemingly unrelated individuals from sarcoglycanopathy cohort and a higher proportion of homozygous markers, thereby indicating common ancestry. Since all these patients are from the south Indian region, we suggest this region to be a primary target of mutation screening in patients diagnosed with sarcoglycanopathy.

Increased DNA strand breaks in spermatozoa of Pxt1 knockout mice.

The Pxt1 gene encodes a male germ cell-specific protein and its overexpression results in male germ cell degeneration and male infertility in transgenic mice. The analysis of the function of Pxt1 during mouse spermatogenesis. The phenotype of Pxt1 knockout mice was characterised by testicular histology, assessment of semen parameters including sperm motility, and DNA fragmentation by flow cytometry. Gene expression was analysed using RT-PCR. Fertility of mutants was checked by standard breeding and competition breeding tests. In Pxt1 -/- mice, a strong increase in the sperm DNA fragmentation index (DFI) was observed, while other sperm parameters were comparable to those of control animals. Despite enhanced DFI, mutants were fertile and able to mate in competition with wild type males. Pxt1 induces cell death; thus, the higher sperm DFI of mice with targeted deletion of Pxt1 suggests some function for this gene in the elimination of male germ cells with chromatin damage. Ablation of mouse Pxt1 results in enhanced DFI. In humans, the homologous PXT1 gene shares 74% similarity with the mouse gene; thus, it can be considered a candidate for mutation screening in patients with increased DFI.

Calreticulin and JAK2 Exon 12 Mutation Screening in Patients with Myeloproliferative Neoplasms’ in Jeddah Region, Saudi Arabia

Background: The JAK2 V617F mutation’s discovery has largely facilitated the comprehension of the myeloproliferative neoplasms’(MPNs) pathogenesis. In recent times, calreticulin (CALR) mutations have been detected in patients with JAK2V617F negative primary myelofibrosis (PMF), and essential thrombocythemia (ET).
 Methods: This study analyzed the impact of JAK 2 Exon 12 and CALR common mutations in 65 patients with JAK2V617F negative MPN from the Jeddah region. An allele-specific polymerase chain reaction (PCR) method was used to screen four common mutations on Exon 12 and direct sequencing and PCR analysis were utilized to screen all patients for CALR.
 Results: None of the patients were positive for the Exon 12 mutation and eight patients were positive for CALR mutations.
 Conclusions: This is the first Saudi Arabian research that focused on screening CALR hotspot mutations and this mutation exists. This fact highlights the importance of implementing diagnostic screening of CALR on MPN patients, in general, and patients with high platelet count, in particular. Further screening of other predisposing genetic markers might facilitate the identification of an important genetic variant, which could aid in the understanding of disease pathogenesis.

Loss of CEP70 function affects acrosome biogenesis and flagella formation during spermiogenesis

The spermatogenesis process is complex and delicate, and any error in a step may cause spermatogenesis arrest and even male infertility. According to our previous transcriptomic data, CEP70 is highly expressed throughout various stages of human spermatogenesis, especially during the meiosis and deformation stages. CEP70 is present in sperm tails and that it exists in centrosomes as revealed by human centrosome proteomics. However, the specific mechanism of this protein in spermatogenesis is still unknown. In this study, we found a heterozygous site of the same mutation on CEP70 through mutation screening of patients with clinical azoospermia. To further verify, we deleted CEP70 in mice and found that it caused abnormal spermatogenesis, leading to male sterility. We found that the knockout of CEP70 did not affect the prophase of meiosis I, but led to male germ-cell apoptosis and abnormal spermiogenesis. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis, we found that the deletion of CEP70 resulted in the abnormal formation of flagella and acrosomes during spermiogenesis. Tandem mass tag (TMT)-labeled quantitative proteomic analysis revealed that the absence of CEP70 led to a significant decrease in the proteins associated with the formation of the flagella, head, and acrosome of sperm, and the microtubule cytoskeleton. Taken together, our results show that CEP70 is essential for acrosome biogenesis and flagella formation during spermiogenesis.

Molecular biological subtypes of breast cancer in BRCA1 mutation carriers

Background. According to the literature, BRCA1-associated breast cancer (BC) most often belongs to the triple negative (TNBC) molecular subtype. The data on the contribution of other molecular subtypes to this group of patients differ among different studies.The study objective is to evaluate the frequency of different tumor molecular subtypes in BC patients with BRCA1 gene mutation treated in N. N. Blokhin National Medical Research Center of Oncology in the period from 2017 to 2020.Materials and methods. The study included BC patients with a mutation in the BRCA1 gene (n = 209) identified as a result BRCA1 mutation screening of patients with BC. DNA diagnostics was carried out on blood samples of patients using the real-time polymerase chain reaction method. After analyzing the patients primary documentation clinical and morphological data were taken into account: the age of diagnosis, the stage of the disease, the results of immunohistochemical studies (estrogen receptor status, progesterone receptor status, HER2 expression, Ki-67 proliferation index). The assignment to the particular molecular tumour subtypes was performed according to estrogen receptor status, progesterone receptor status, HER2 status and Ki67 value.Results. Clinical and pathomorphological data of 209 patients with BRCA1-associated BC were analyzed. The age at diagnosis ranged from 23 to 72 years, the median age was 40 years, the mean age was 41.46 ± 9.82 years. BC associated with BRCA1 was found to be TNBC in 71.3 % and luminal B, HER2 negative (LumB–) in 19.1 % of the cases. Other tumour subtypes were much less common: luminal B, HER2 positive (LumB+) in 7.2 %, luminal A (LumA) in 1 % and HER2-positive (HER2+) in 1.4 % of the cases. The frequency of subtypes was estimated in different age groups (1st – patients 23–34 (n = 53), 2nd – 35–49 (n = 111), and 3rd – 50–72 (n = 45) years old). TNBC frequency was 81.1 % in the 1st group, 73.9 % in the 2nd and 53.4 % in the 3rd group; LumB– frequency was 15.1, 15.3 and 33.3 % respectively. Using the Fisher test it was shown that the differences in frequencies were statistically significant between groups 1st and 3 rd, as well as between groups 2 nd and 3 rd (p <0.05).Conclusion. TNBC was the main molecular subtype in all age groups of BC patients with BRCA1 germinal mutation, TNBC frequency was lower in the older age group. LumB– subtype was also common in BRCA1-associated tumors especially in older women.

Screening for genetic mutations in patients with neuropathy without definite etiology is useful

To determine the clinical usefulness of systemic genetic testing in neuropathies without definite etiology. We systematically performed genetic testing in all patients with neuropathy who did not have a definite etiology, seen at our neuromuscular clinic between 2017 and 2020. The testing consisted of an inherited neuropathy panel (72-81 genes), which used next-generation sequencing technology. We screened 200 patients. Pathogenic mutations were found in 30 (15%). PMP22, TTR and GJB1, accounted for 83.3% of positive mutations. The management was altered in four patients (2%). Two patients were found to have hereditary transthyretin amyloidosis and were started on TTR gene silencers. Two patients were being treated for demyelinating autoimmune neuropathy and were diagnosed with CMT1A and CMTX. Screening for genetic mutations in patients with neuropathy without a definite etiology is useful. While only a minority of patients with unsuspected inherited neuropathy tested positive, the findings altered management in some, improving morbidity and, perhaps, mortality.

Genome-Scale Analysis Identifies Novel Genetic Vulnerabilities in Allan Herndon Dudley Syndrome

Allan Herndon Dudley syndrome (AHDS), an X-linked condition, constitutes a rare, progressive, clinically heterogeneous disorder involving severe psychomotor retardation and peripheral thyrotoxicosis, consequent to thyroid hormone transporter SLC16A2 mutations. However, direct targeted screening for SLC16A2 mutations in patients with AHDS partly predicts phenotypic heterogeneity although similar mutations are associated with differences in clinical phenotype severity, suggesting the existence of additional gene-related AHDS subtypes as in other types of mental retardation. Specifically, we report a family with AHDS comprising five individuals, including three male children (of whom two were monozygotic twins). In this pedigree, we identified a linkage of two X-linked inherited genes including SLC16A2 (p. G453R) and KIF4A (p. A491V) as a putative AHDS etiology. Additionally, we identified an independent cluster of structural variations (SVs)-enriched potential AHDS susceptibility genes, which might be as the neuronal genetic vulnerabilities likely contributing to AHDS. Our observations suggest that AHDS phenotype heterogeneity may arise from multi-genetic contributions. These familial cases, involving likely novel AHDS subtypes, provide an opportunity to explain neuropsychiatric retardation. An integrated next generation mapping and whole genome sequencing strategy for refining AHDS diagnosis and clinical categorization is recommended inclusive of cases with the related overlapping phenotypes in psychomotor disabilities. Funding Statement: This work was supported by the Stem Cell Clinical Trial Project of the National Health and Family Planning Commission of China and the China Food and Drug Administration (Registration No. CMR-201611-1003), (Medical Research Registry and Management Systemhttp://114.255.123.14), and the National Nature Science Foundation of China (Grant No. 81471308). Declaration of Interests: We declare no conflict of interest. Ethics Approval Statement: Informed consent was obtained from the parents, and the study was approved by the Research Ethics Boards at the First Affiliated Hospital of Dalian Medical University (approval number: YJ-KY-FB-2017-27)

DNAJC13 mutation screening in patients with Parkinson's disease from South Italy

BackgroundParkinson's disease (PD) is the second most common neurodegenerative disorder, and the most common neurodegenerative form of parkinsonism. Recently, a pathogenic mutation (p.N855S) in DNAJC13 was linked to autosomal dominant Lewy body PD in a Dutch–German–Russian Mennonite multi-incident kindred, and was found in five additional patients. In this study, we performed a comprehensive screening of the DNAJC13 gene in familial PD and sporadic PD to assess the frequency of known and novel rare nonsynonymous variants. MethodsWe screened 563 sporadic and 168 familial PD patients and a control series (n = 1000) for the coding region of DNAJC13. ResultsOur sequencing analysis identified two carriers of the c.2708G > A (p.R903K) variant in exon 24 of DNAJC13. One of these carriers is a familial PD. ConclusionThe p. R903K variant was not found in 1000 healthy controls and it is localized in a functional domain of the DNAJC13 protein. Further studies are necessary to evaluate the role of DNAJC13 variants in PD.

Significant prevalence of NR3C1 mutations in incidentally discovered bilateral adrenal hyperplasia: results of the French MUTA-GR Study

Recently discovered mutations of NR3C1 gene, encoding for the GR, in patients with glucocorticoid resistance and bilateral adrenal incidentalomas prompted us to investigate whether GR mutations might be associated with adrenal hyperplasia. The multicenter French Clinical Research Program (Muta-GR) was set up to determine the prevalence of GR mutations and polymorphisms in patients harboring bilateral adrenal incidentalomas associated with hypertension and/or biological hypercortisolism without clinical Cushing's signs. One hundred patients were included in whom NR3C1 sequencing revealed five original heterozygous GR mutations that impaired GR signaling in vitro. Mutated patients presented with mild glucocorticoid resistance defined as elevated urinary free cortisol (1.7 ± 0.7 vs 0.9 ± 0.8 upper limit of normal range, P = 0.006), incomplete 1 mg dexamethasone suppression test without suppressed 8-AM adrenocorticotrophin levels (30.9 ± 31.2 vs 16.2 ± 17.5 pg/mL) compared to the non-mutated patients. Potassium and aldosterone levels were lower in mutated patients (3.6 ± 0.2 vs 4.1 ± 0.5 mmol/L, P = 0.01, and 17.3 ± 9.9 vs 98.6 ± 115.4 pg/mL, P = 0.0011, respectively) without elevated renin levels, consistent with pseudohypermineralocorticism. Ex vivo characterization of mutated patients' fibroblasts demonstrated GR haploinsufficiency as revealed by below-normal glucocorticoid induction of FKBP5 gene expression. There was no association between GR polymorphisms and adrenal hyperplasia in this cohort, except an over-representation of BclI polymorphism. The 5% prevalence of heterozygous NR3C1 mutations discovered in our series is higher than initially thought and encourages GR mutation screening in patients with adrenal incidentalomas to unambiguously differentiate from Cushing's states and to optimize personalized follow-up.

VSX1 and SOD1 Mutation Screening in Patients with Keratoconus.

VSX1 and SOD1 Mutation Screening in Patients with Keratoconus.

P.143 - POPDC1 gene mutation screening in patients with LGMD and heart disturbances: a mutation load effect?

POPDC1 is the best-studied member of the Popeye domain-containing gene family. The members of this family encode transmembrane proteins, which are abundantly expressed in cardiac and skeletal muscle. The evolutionary conserved Popeye domain functions as a high-affinity 3'-5'-cyclic adenosine monophosphate (cAMP) binding domain. Recently, we identified recessive POPDC1 mutations in a family affected by Limb-girdle Muscular Dystrophy and atrioventricular (AV) block (OMIM LGMD2X). In order to evaluate if POPDC1 may contribute to phenotype variability in other LGMDs with or without cardiac involvement we screened by Sanger sequencing 166 patients with different phenotypes either orphan of gene mutations or with known gene mutations. We identified 8 different heterozygous POPDC1 mutations in 10 patients, 6 of them do also carry LMNA mutations, and 2 patients do carry Dysferlin mutations. Phenotypes vary from classical LGMD phenotype to pure cardiomyopathy with rhythm disturbances. It is emerging that the mutation load (occurrence of multiple mutations in different causative genes) plays a relevant role in phenotype modulation. We suggest that POPDC1 gene variations may contribute to the high clinical heterogeneity and intra- and interfamilial phenotypic variability of LMNA diseases. This hypothesis is supported by the known functional interaction between POPDC1 and LMNA.

Improving Mutation Screening in Patients with Colorectal Cancer Predisposition Using Next-Generation Sequencing

Identification of genetic alterations is important for family risk assessment in colorectal cancers. Next-generation sequencing (NGS) technologies provide useful tools for single-nucleotide and copy number variation (CNV) identification in many genes and samples simultaneously. Herein, we present the validation of current Multiplicom MASTR designs of mismatch repair combined to familial adenomatous polyposis genes in a single PCR reamplification test for eight DNA samples simultaneously on a MiSeq apparatus. Blood samples obtained from 224 patients were analyzed. We correctly identified the 97 mutations selected among 48 samples tested in a validation cohort. PMS2 NGS analysis of the eight positive controls identified single-nucleotide variations not detected with targeted referent methods. As NGS method could not discriminate if some of them were assigned to PMS2 or pseudogenes, only CNV analysis with multiplex ligand probe-dependent amplification confirmation was retained for clinical use. Twenty-seven new variants of unknown significance, 21 disease-causing variants, and two CNVs were detected among the 176 patient samples analyzed in diagnosis routine. MUTYH disease-causing mutations were identified in two patient samples assessed for mismatch repair testing, confirming that this method facilitates accurate and rapid individual risk assessments. In one sample, the MUTYH mutation was associated with a MSH6 disease-causing mutation, suggesting that this method is helpful to identify additional cancer risk modifiers and provides a useful tool to optimize clinical issues.

VSX1 and SOD1 Mutation Screening in Patients with Keratoconus in the South of Iran.

Purpose:To investigate mutations of visual system homeobox 1 (VSX1) and superoxide dismutase 1 (SOD1) in 20 patients with keratoconus in the south of Iran.Methods:Twenty patients with keratoconus who had a positive familial history were enrolled in this study and gave informed consent for DNA analysis. Genomic DNA was extracted from peripheral blood lymphocytes. Polymerase chain reaction (PCR) was carried out to amplify exon 2 of SOD1 and its exon-intron boundary for the detection of a seven-base deletion in intron 2 of SOD1, and also all five exons of VSX1 and their exon-intron boundaries. Amplified samples were then subjected to direct DNA sequencing.Results:Sequencing data were compared against reference sequences using NCBI basic local alignment search tool (BLAST), which revealed that our patients had no mutations in SOD1 and VSX1. Two single-nucleotide polymorphisms (SNPs), namely in VSX1(rs58752432 and rs59089167) were found in six patients.Conclusion:Mutations in VSX1 and SOD1 genes associated with keratoconus were not identified in our patients. Therefore, it will be necessary to investigate other chromosomal loci for potential causal mutations of keratoconus using next generation sequencing (NGS) methods in our population.

The COQ2 genotype predicts the severity of coenzyme Q10 deficiency.

COQ2 (p-hydroxybenzoate polyprenyl transferase) encodes the enzyme required for the second step of the final reaction sequence of Coenzyme Q10 (CoQ) biosynthesis. Its mutations represent a frequent cause of primary CoQ deficiency and have been associated with the widest clinical spectrum, ranging from fatal neonatal multisystemic disease to late-onset encephalopathy. However, the reasons of this variability are still unknown.We have characterized the structure of human COQ2, defined its subcellular localization and developed a yeast model to validate all the mutant alleles reported so far.Our findings show that the main functional transcript of COQ2 is shorter than what was previously reported and that its protein product localizes to mitochondria with the C-terminus facing the intermembrane space. Complementation experiments in yeast showed that the residual activity of the mutant proteins correlates with the clinical phenotypes observed in patients.We defined the structure of COQ2 with relevant implications for mutation screening in patients and demonstrated that, contrary to other COQ gene defects such as ADCK3, there is a correlation between COQ2 genotype and patient's phenotype.

The association between brain natriuretic peptide and tissue Doppler parameters in children with hypertrophic cardiomyopathy.

In this study, we investigated the association between brain natriuretic peptide (BNP) levels and tissue Doppler imaging measurements and also screening for deadly mutations in patients with hypertrophic cardiomyopathy (HCM). We enrolled 20 patients diagnosed with HCM (age:10.7±5 years (1-17), 85% male, weight:42.25±23.10 kg, height:141.80±32.45 cm) and 20 age, gender and body weight-matched control subjects. We performed electrocardiography, transthoracic echocardiography, and tissue Doppler echocardiography in each group, as well as genetic tests (for Arg403Gln, Arg453Cys, Arg719Trp and Arg719Gln mutations in MYH7 Exons 13, 14, 19) and BNP in the patients. The patients were divided into two groups according to the presence (Group 1) or absence (Group 2) of left ventricular (LV) outflow tract obstruction. QTc dispersion and the LV ejection fraction and left atrial (LA) volume index were increased in Group 1. The LA volume index and the mitral and septal E/Ea ratio and septum Z-score were increased while the mitral lateral annulus and septal annulus Ea wave velocities and the mitral and tricuspid E/A ratio were decreased in patients with high levels of BNP compared to those with normal BNP levels. There were no mutations that are associated with increased risk of sudden death found in patients included in this study. In the light of our data, we conclude that such parameters BNP levels above the 98 pg/mL, septal thickness Z-score ˃6, and higher mitral and septal E/Ea ratios can be used for management of patients with HCM according to life-threatening conditions.

Prevalence and Prognostic Impact of CEBPA Gene Mutation (Simplified Assay Technique) in Egyptian Acute Myeloid Leukemia Patients with Normal Cytogenetics.

Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), especially in those with a normal cytogenetics. However, few studies were done on Egyptian AML patients and none of them look for easier and less expensive method for CEBPA mutation screening. This study is aimed to investigate the prevalence of CEBPA mutations and its clinical and prognostic impact in Egyptian patients with cytogenetically normal AML (CN-AML). This was done using fragment analysis to assess this method as a cheaper and less laborious screening method compared to sequencing. Fluorescent PCR was done to amplify CEBPA gene in DNA extracted from 40 CN-AML patients. This was followed by fragment analysis of post-PCR products using GeneMapper software for detection of CEBPA mutations. CEBPA gene mutations were found in 7/40 CN-AML patients (17.5%) and it was significantly associated with lower LDH levels (p=0.039). All patients with CEBPA mutations achieved clinical remission and none of them showed refractoriness, relapsed, or died by the end of the 2years study period. Furthermore, those patients demonstrate significantly longer overall and disease free survival than those with wild type CEBPA gene (p=0.001 and 0.004 respectively). CEBPA mutation has a favorable prognostic impact in CN-AML. Fragment analysis is a good, lees laborious and cheaper method that can be used for CEBPA mutation screening in patients with CN-AML.

ISPD mutations account for a small proportion of Italian Limb Girdle Muscular Dystrophy cases.

BackgroundLimb Girdle Muscular Dystrophy (LGMD), caused by defective α-dystroglycan (α-DG) glycosylation, was recently associated with mutations in Isoprenoid synthase domain-containing (ISPD) and GDP-mannose pyrophosphorylase B (GMPPB) genes. The frequency of ISPD and GMPPB gene mutations in the LGMD population is unknown.MethodsWe investigated the contributions of ISPD and GMPPB genes in a cohort of 174 Italian patients with LGMD, including 140 independent probands. Forty-one patients (39 probands) from this cohort had not been genetically diagnosed. The contributions of ISPD and GMPPB were estimated by sequential α-DG immunohistochemistry (IHC) and mutation screening in patients with documented α-DG defect, or by direct DNA sequencing of both genes when muscle tissue was unavailable.ResultsWe performed α-DG IHC in 27/39 undiagnosed probands: 24 subjects had normal α-DG expression, two had a partial deficiency, and one exhibited a complete absence of signal. Direct sequencing of ISPD and GMPPB revealed two heterozygous ISPD mutations in the individual who lacked α-DG IHC signal: c.836-5 T > G (which led to the deletion of exon 6 and the production of an out-of-frame transcript) and c.676 T > C (p.Tyr226His). This patient presented with sural hypertrophy and tip-toed walking at 5 years, developed moderate proximal weakness, and was fully ambulant at 42 years. The remaining 12/39 probands did not exhibit pathogenic sequence variation in either gene.ConclusionISPD mutations are a rare cause of LGMD in the Italian population, accounting for less than 1 % of the entire cohort studied (FKRP mutations represent 10 %), while GMPPB mutations are notably absent in this patient sample. These data suggest that the genetic heterogeneity of LGMD with and without α-DG defects is greater than previously realized.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-015-0428-8) contains supplementary material, which is available to authorized users.

RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling.

Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p.(Pr270Leu), p.(Ala756Val) and p.(Tyr1062Cys) resulted in lower activation of RET. Moreover, only two RET variants (p.(Ala756Val) and p.(Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A>G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling.

BRAF mutation screening in melanoma: is sentinel lymph node reliable?

As the detection of the BRAF V600E mutation has a direct impact on treatment decision, an accurate screening for BRAF mutations in patients with advanced or metastatic melanoma is mandatory. Nevertheless, BRAF oncogene mutation status between different samples from the same patient has been studied with conflicting results. This study investigated the intrapatient homogeneity of BRAF mutation status using pyrosequencing in primary tumors and different metastatic sites of melanoma patients. Paired samples of lymphatic, visceral, and subcutaneous metastases and primary melanoma from 45 metastatic melanoma patients were tested for BRAF mutations using a pyrosequencing assay and by Sanger sequencing. Overall, sequencing for BRAF mutation status was performed in 114 paired samples from 45 patients. Eighteen patients (40%) carried a BRAF mutation, including BRAF V600E (12/18), BRAF V600K (5/18), and BRAF V600R (1/18) mutations. Multiple BRAF mutations (V600E and V600K) were found in one patient. Among the patients with BRAF mutations, a good agreement in BRAF mutation status was found between the first and second tumor samples genotyped (91%; Cohen's κ coefficient: 0.81). Discordance in BRAF mutation status was found only in four patients, involving all three patients in whom sentinel lymph node (SLN) metastases were sampled. These SLNs exhibited a wild-type genotype and were discordant with the other BRAF-mutated samples found in the same patient. The intrapatient BRAF status was predominantly homogeneous. However, SLN genotyping using pyrosequencing might be inaccurate in determining the actual mutation status of melanoma. Further studies are required to confirm the lack of reliability of SLN.

SNPscan as a high-performance screening tool for mutation hotspots of hearing loss-associated genes

In the present study, to assess the feasibility of the SNPscan technique for mutation screening in patients with nonsyndromic hearing loss (NSHL) and neonatus in China, the SNPscan technique was compared with the SNaPshot screening system. Chinese patients (162) with NSHL were used as the experimental group and 276 children without HL were used as the control group, respectively. SNPscan detected molecular defects in 112 patients (68.5%). In this technique, 83 patients (51.2%) with homozygous or compound heterozygous had confirmed molecular etiology in the GJB2, SLC26A4, and MT-RNR1 genes. By contrast, SNaPshot detected molecular defects in 103 patients (63.6%). In this method, 72 subjects (44.4%) with HL were confirmed to have NSHL caused by these mutations. This study demonstrates that SNPscan performs equally well or better than earlier routine genotyping method for genetic hearing loss, with possibility of detecting a larger variety of mutation.