Mutation analysis of the <Emphasis Type="Italic">COL1A1</Emphasis> and <Emphasis Type="Italic">COL1A2</Emphasis> genes in Vietnamese patients with osteogenesis imperfecta

Background

The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI.

Methods

Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish’s osteogenesis imperfecta mutation database.

Results

The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G?>?A (p.Gly821Ser) in four unrelated patients and one, c.2005G?>?A (p.Ala669Thr), in two unrelated patients.

Conclusion

Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

     

Identification of a Novel Mutation in the COL2A1 Gene in a Chinese Family with Spondyloepiphyseal Dysplasia Congenita

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant chondrodysplasia characterized by disproportionate short-trunk dwarfism, skeletal and vertebral deformities. Exome sequencing and Sanger sequencing were performed in a Chinese Han family with typical SEDC, and a novel mutation, c.620G>A (p.Gly207Glu), in the collagen type II alpha-1 gene (COL2A1) was identified. The mutation may impair protein stability, and lead to dysfunction of type II collagen. Family-based study suggested that the mutation is a de novo mutation. Our study extends the mutation spectrum of SEDC and confirms genotype-phenotype relationship between mutations at glycine in the triple helix of the alpha-1(II) chains of the COL2A1 and clinical findings of SEDC, which may be helpful in the genetic counseling of patients with SEDC.     

Hearing impairment in Stickler syndrome: a systematic review

Background

Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely COL2A1, COL11A1 and COL11A2 (autosomal dominant inheritance), and COL9A1 and COL9A2 (autosomal recessive inheritance). The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype.

Methods

English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation.

Results

313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%).

Conclusions

Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired.

     

Mutational analysis of COL1A1 and COL1A2 genes among Estonian osteogenesis imperfecta patients

Background

Osteogenesis imperfecta (OI) is a rare bone disorder. In 90% of cases, OI is caused by mutations in the COL1A1/2 genes, which code procollagen α1 and α2 chains. The main aim of the current research was to identify the mutational spectrum of COL1A1/2 genes in Estonian patients. The small population size of Estonia provides a unique chance to explore the collagen I mutational profile of 100% of OI families in the country.

Methods

We performed mutational analysis of peripheral blood gDNA of 30 unrelated Estonian OI patients using Sanger sequencing of COL1A1 and COL1A2 genes, including all intron-exon junctions and 5′UTR and 3′UTR regions, to identify causative OI mutations.

Results

We identified COL1A1/2 mutations in 86.67% of patients (26/30). 76.92% of discovered mutations were located in the COL1A1 (n = 20) and 23.08% in the COL1A2 (n = 6) gene. Half of the COL1A1/2 mutations appeared to be novel. The percentage of quantitative COL1A1/2 mutations was 69.23%. Glycine substitution with serine was the most prevalent among missense mutations. All qualitative mutations were situated in the chain domain of pro-α1/2 chains.

Conclusion

Our study shows that among the Estonian OI population, the range of collagen I mutations is quite high, which agrees with other described OI cohorts of Northern Europe. The Estonian OI cohort differs due to the high number of quantitative variants and simple missense variants, which are mostly Gly to Ser substitutions and do not extend the chain domain of COL1A1/2 products.

     

Modification of optimal pH in l-arabinose isomerase from Geobacillus stearothermophilus for d-galactose isomerization

l-Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d-galactose, which is not a natural substrate. To change the optimal pH of GSAI for d-galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val⁴⁰⁸ and Asn⁴⁷⁵ were determined to be highly influential mutation points for d-galactose isomerization activity. A random mutation was introduced into sites Val⁴⁰⁸ and Asn⁴⁷⁵ (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d-galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively.     

Specificity of recA441-mediated (tif-1) mutational events.

Summary To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI ^– mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G : C C : G, G : C T : A and A : T T : A transversion events were specifically enhanced after SOS induction. A preferential 5-Y-Purine-3 neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5-C/GTGG-3 sequences is also observed. Fifty events were recovered at the lacI frameshift hotspot site and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA⁺ background and is a consequence of the fourfold induction of the (–)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI ^– mutations.     

Molecular characterization of sessile serrated adenoma/polyps with dysplasia/carcinoma based on immunohistochemistry,next-generation sequencing,and microsatellite instability testing: a case series study

Background

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are considered early precursor lesions in the serrated neoplasia pathway. Recent studies have shown associations of SSA/Ps with lost MLH1 expression, a CpG island methylator phenotype, and BRAF mutations. However, the molecular biological features of SSA/Ps with early neoplastic progression have not yet been fully elucidated, owing to the rarity of cases of SSA/P with advanced histology such as cytologic dysplasia or invasive carcinoma. In this study, we aimed to elucidate the molecular biological features of SSA/Ps with dysplasia/carcinoma, representing relatively early stages of the serrated neoplasia pathway.

Methods

We performed immunostaining for β-catenin, MLH1, and mucins (e.g., MUC2, MUC5AC, MUC6, and CD10); targeted next-generation sequencing; and microsatellite instability (MSI) testing in 8 SSA/P lesions comprised of 4 SSA/Ps with high-grade dysplasia and 4 SSA/Ps with submucosal carcinoma.

Results

Lost MLH1 expression was found in 5 cases. All lesions studied were positive for nuclear β-catenin expression. Regarding phenotypic mucin expression, all lesions were positive for MUC2, but negative for CD10. MUC5AC and MUC6 positivity was observed in 7 cases. Genetically, the most frequently mutated gene was BRAF (7 cases), and other mutations were detected in FBXW7 (3 cases); TP53 (2 cases), and KIT, PTEN, SMAD4, and SMARCB1 (1 case each). Furthermore, 4 of 8 lesions were MSI-high and the remaining 4 lesions were microsatellite-stable (MSS). Interestingly, all 4 MSI-high lesions displayed MLH1 loss, 3 of which harbored a FBXW7 mutation, but not a TP53 mutation. However, 2 MSS lesions harbored a TP53 mutation, although none harbored a FBXW7 mutation.

Conclusions

SSA/Ps with dysplasia/carcinoma frequently harbored BRAF mutations. Activation of the WNT/β-catenin signaling pathway may facilitate the development of dysplasia in SSA/Ps and progression to carcinoma. Furthermore, our results suggested that these lesions might be associated with both MSI-high and MSS colorectal cancer, which might be distinguished by distinct molecular biological features such as lost MLH1 expression, FBXW7 mutations, and TP53 mutations.

     

Whole-exome sequencing identifies novel pathogenic mutations and putative phenotype-influencing variants in Polish limb-girdle muscular dystrophy patients

Background

Limb girdle muscular dystrophies (LGMD) are a group of heterogeneous hereditary myopathies with similar clinical symptoms. Disease onset and progression are highly variable, with an elusive genetic background, and around 50% cases lacking molecular diagnosis.

Methods

Whole exome sequencing (WES) was performed in 73 patients with clinically diagnosed LGMD. A filtering strategy aimed at identification of variants related to the disease included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of genes expressed in muscle, analysis of the disease-associated interactome and copy number variants analysis.

Results

Genetic diagnosis was possible in 68.5% of cases. On average, 36.3 rare variants in genes associated with various muscle diseases per patient were found that could relate to the clinical phenotype. The putative causative mutations were mostly in LGMD-associated genes, but also in genes not included in the current LGMD classification (DMD, COL6A2, and COL6A3). In three patients, mutations in two genes were suggested as the joint cause of the disease (CAPN3+MYH7, COL6A3+CACNA1S, DYSF+MYH7). Moreover, a variety of phenotype-influencing variants were postulated, including in patients with an identified already known primary pathogenic mutation.

Conclusions

We hypothesize that LGMD could be better described as oligogenic disorders in which dominant clinical presentation can result from the combined effect of mutations in a set of genes. In this view, the inter- and intrafamilial variability could reflect a specific genetic background and the presence of sets of phenotype-influencing or co-causative mutations in genes that either interact with the known LGMD-associated genes or are a part of the same pathways or structures.

     

Combined genetic mutations have remarkable effect on deep venous thrombosis and/or pulmonary embolism occurence

Purpose

Although deep vein thrombosis and thromboembolic diseases differ among various races, they are still important in our day. The difficulties in treatment and following-up of these diseases are caused by secret genetic mutations rather than predisposing factors.

Methods

Between January 2011 and May 2013, patients who were traced for deep vein thrombosis and/or pulmonary embolism were evaluated retrospectively. 84 patients (53.6% males and 46.4% females) were included in the study. Their family histories, predisposing factors and treatments were researched. Factor V Leiden (G 1691A), Factor II G20210A, Plasminogen Activator Inhibitor-Type 1 (4G/5G), and Methylene Tetrahydrofolate Reductase (C677T, A1298C) mutations were investigated from peripheral venous blood.

Results

Among the genetic mutations we searched, the incidence of single mutation rate was observed at 11.9%, double mutation collocation at 44%, triple mutation collocation at 29.8%, quadruple mutation collocation at 13.1%, and finally, quintuplet mutation collocation at 1.2%. Our approximate mutation number was found as 2.47 ± 0.91.

Conclusion

We observed that multiple mutations were high in number compared to single genetic mutations. The patients who have multiple mutations should be more in the front line considering their diagnosis, treatment and following up, and also in terms of decreasing mortality, morbidity and recurrence.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test

Background

Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.

Methodology/Principal Findings

A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.

Conclusions/Significance

The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.     

Abundance,Distribution, and Mutation Rates of Homopolymeric Nucleotide Runs in the Genome of Caenorhabditis elegans

Homopolymeric nucleotide runs, also called mononucleotide microsatellites, are a ubiquitous, dominant, and mutagenic feature of eukaryotic genomes. A clear understanding of the forces that shape patterns of homopolymer evolution, however, is lacking. We provide a focused investigation of the abundance, chromosomal distribution, and mutation spectra of the four strand-specific homopolymer types (A, T, G, C) 8 bp in the genome of Caenorhabditis elegans. A and T homopolymers vastly outnumber G and C HPs, and the run-length distributions of A and T homopolymers differ significantly from G and C homopolymers. A scanning window analysis of homopolymer chromosomal distribution reveals distinct clusters of homopolymer density in autosome arms that are regions of high recombination in C. elegans. Dramatic biases are detected among closely spaced homopolymers; for instance, we observe 994 A homopolymers immediately followed by a T homopolymer (5 to 3) and only 8 instances of T homopolymers directly followed by an A homopolymer. Empirical homopolymer mutation assays in a set of C. elegans mutation-accumulation lines reveal an 20-fold higher mutation rate for G and C homopolymers compared to A and T homopolymers. Nuclear A and T homopolymers are also found to mutate 100-fold more slowly than mitochondrial A and T homopolymers. This integrative approach yields a total nuclear genome-wide homopolymer mutation rate estimate of 1.6 mutations per genome per generation.Novel sequences are deposited in GenBank under accession numbers AY219759–AY219789.     

A novel mutation screening system for Ehlers-Danlos Syndrome, vascular type by high-resolution melting curve analysis in combination with small amplicon genotyping using genomic DNA

Ehlers-Danlos syndrome, vascular type (vEDS) (MIM #130050) is an autosomal dominant disorder caused by type III procollagen gene (COL3A1) mutations. Most COL3A1 mutations are detected by using total RNA from patient-derived fibroblasts, which requires an invasive skin biopsy. High-resolution melting curve analysis (hrMCA) has recently been developed as a post-PCR mutation scanning method which enables simple, rapid, cost-effective, and highly sensitive mutation screening of large genes. We established a hrMCA method to screen for COL3A1 mutations using genomic DNA. PCR primers pairs for COL3A1 (52 amplicons) were designed to cover all coding regions of the 52 exons, including the splicing sites. We used 15 DNA samples (8 validation samples and 7 samples of clinically suspected vEDS patients) in this study. The eight known COL3A1 mutations in validation samples were all successfully detected by the hrMCA. In addition, we identified five novel COL3A1 mutations, including one deletion (c.2187delA) and one nonsense mutation (c.2992C>T) that could not be determined by the conventional total RNA method. Furthermore, we established a small amplicon genotyping (SAG) method for detecting three high frequency coding-region SNPs (rs1800255:G>A, rs1801184:T>C, and rs2271683:A>G) in COL3A1 to differentiate mutations before sequencing. The use of hrMCA in combination with SAG from genomic DNA enables rapid detection of COL3A1 mutations with high efficiency and specificity. A better understanding of the genotype–phenotype correlation in COL3A1 using this method will lead to improve in diagnosis and treatment.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

Genetic analysis of <Emphasis Type="Italic">COL11A2</Emphasis> in Korean patients with autosomal dominant non-syndromic hearing loss

The collagen type XI alpha 2 gene (COL11A2) is associated with autosomal dominant non-syndromic hearing loss (ADNSHL), and all mutations of this gene in ADNSHL are missense mutations. To evaluate its potential as a major causative gene of ADNSHL in the Korean population, we performed genetic analysis of COL11A2 in 75 unrelated Korean patients with ADNSHL. Consequently, 5 non-synonymous variants, 7 synonymous variants, and 6 intronic variants were identified in COL11A2. Among them, a novel variant, p.G829R (c.2485G>C) was found in a patient as a heterozygote. However, pedigree analysis showed this variation was not co-segregated with hearing loss. Previously reported variants p.G230W (c.688G>T) and p.P1422L (c.4265C>T) were discovered in Korean patients. However, these variants were also detected in normal individuals. These results suggest that COL11A2 is not a major causative gene of ADNSHL in the Korean population.     

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Jeddah,Kingdom of Saudi Arabia

Background

The development of polymerase chain reaction (PCR)-based methods for the detection of known mutations has facilitated detecting specific red blood cell (RBC) enzyme deficiencies. We carried out a study on glucose-6-phosphate dehydrogenase (G6PD) deficient subjects in Jeddah to evaluate the molecular characteristics of this enzyme deficiency and the frequency of nucleotide1311 and IVS-XI-93 polymorphisms in the glucose-6-phosphate dehydrogenase gene.

Results

A total of 1584 unrelated Saudis (984 neonates and 600 adults) were screened for glucose-6-phosphate dehydrogenase deficiency. The prevalence of glucose-6-phosphate dehydrogenase deficiency was 6.9% (n = 110). G6PD Mediterranean mutation was observed in 98 (89.1%) cases, G6PD Aures in 11 (10.0%) cases, and G6PD Chatham in 1 (0.9%) case. None of the samples showed G6PD A￣ mutation. Samples from 29 deficient subjects (25 males and 4 females) were examined for polymorphism. The association of two polymorphisms of exon/intron 11 (c.1311T/IVS-XI-93C) was observed in 14 (42.4%) of 33 chromosomes studied. This association was found in 9 (31.0%) carriers of G6PD Mediterranean and in 4 (13.8%) carriers of G6PD Aures.

Conclusions

The majority of mutations were G6PD Mediterranean, followed by G6PD Aures and < 1% G6PD Chatham. We conclude that 1311T is a frequent polymorphism in subjects with G6PD Mediterranean and Aures variants in Jeddah.

     

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients

Background

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing.

Results

Our custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing.

Conclusions

NGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.

     

Low frequency of BRAF and KRAS mutations in Chinese patients with low-grade serous carcinoma of the ovary

Background

Mounting evidence has shown that KRAS and BRAF are somatic mutations associated with low grade serous carcinoma (LGSC) of the ovary. However, the frequency of KRAS or BRAF mutation was variable in literatures, with a frequency of 16–54% for KRAS mutation and 2–33% for BRAF mutation. Meanwhile, the prognostic significance of KRAS or BRAF mutation remains controversial.

Methods

Codons 12 and 13 of exon 2 of KRAS gene and exon 15 of BRAF gene were analyzed using direct Sanger sequencing in 32 cases of LGSC of the ovary. The associations between KRAS or BRAF mutation and clinicopathological characteristics, overall survival (OS) and disease-free survival (DFS) were statistically analyzed.

Results

KRAS mutation was observed in nine cases (9/32, 28%) and BRAF mutation in two cases (2/32, 6%). KRAS and BRAF mutations were mutually exclusive. Neither KRAS nor BRAF mutation was statistically associated with OS or DFS in our cohort, although there was a favorable prognostic trend in patients with KRAS G12D mutation than those with KRAS G12 V mutation or wild-type KRAS for OS.

Conclusions

The present study indicated a low frequency of BRAF or KRAS mutation in Chinese patients with LGSC of the ovary, and neither KRAS nor BRAF mutation is a prognostic factor.

     

A novel mutation (R122Q) in the cathepsin K gene in a Chinese child with Pyknodysostosis

Background

Pyknodysostosis (OMIM 265800) is a rare, autosomal recessive sclerosing skeletal dysplasia as a consequence of the diminished capacity of osteoclasts to degrade organic bone matrix. Pyknodysostosis is caused by mutation in the cathepsin K (CTSK) gene. Up to date, 34 different CTSK mutations have been identified in patients with Pyknodysostosis; however, only one mutation was previously identified in a Chinese patient. The objective of this study was to characterize the clinical manifestations and features of Pyknodysostosis and identify the mutation of the causative gene in a Chinese family with Pyknodysostosis.

Methods

We investigated a non-consanguineous Chinese family in which an 11-year-old child was affected with Pyknodysostosis. Altogether, 203 persons, including the affected individual, his parents and 200 healthy donors, were recruited and genomic DNA was extracted. All 8 exons of the CTSK gene, including the exon–intron boundaries, were amplified and sequenced directly.

Results

The proband displayed a novel homozygous missense mutation c.365G>A in exon 4 of the CTSK gene. This mutation leads to the substitution of the arginine at position 122 by glutamine (R122Q) in cathepsin K. The parents were heterozygous for this gene mutation, and the mutation was not found in the 200 unrelated controls.

Conclusion

Our study suggests that the novel missense mutation c.365G>A (R122Q) in exon 4 of CTSK gene was responsible for Pyknodysostosis in the Chinese family.