NDUFV1 mutations in complex I deficiency: Case reports and review of symptoms

Mitochondrial complex I (CI) deficiency is the most common oxidative phosphorylation disorder described. It shows a wide range of phenotypes with poor correlation within genotypes. Herein we expand the clinics and genetics of CI deficiency in the brazilian population by reporting three patients with pathogenic (c.640G>A, c.1268C>T, c.1207dupG) and likely pathogenic (c.766C>T) variants in the NDUFV1 gene. We show the mutation c.766C>T associated with a childhood onset phenotype of hypotonia, muscle weakness, psychomotor regression, lethargy, dysphagia, and strabismus. Additionally, this mutation was found to be associated with headaches and exercise intolerance in adulthood. We also review reported pathogenic variants in NDUFV1 highlighting the wide phenotypic heterogeneity in CI deficiency.     

Hsa-miR-34b/c rs4938723 T>C and hsa-miR-423 rs6505162 C>A Polymorphisms Are Associated with the Risk of Esophageal Cancer in a Chinese Population

Esophageal cancer is the eighth most common cancer and sixth leading cause of cancer associated death worldwide. Besides environmental risk factors, genetic factors might play an important role in the esophageal cancer carcinogenesis. We conducted a hospital based case–control study to evaluate the genetic susceptibility of functional single nucleotide polymorphisms (SNPs) in the microRNAs on the development of esophageal cancer. A total of 629 esophageal squamous cell carcinoma (ESCC) cases and 686 controls were recruited for this study. The hsa-miR-34b/c rs4938723 T>C, pri-miR-124-1 rs531564 C>G, pre-miR-125a rs12975333 G>T and hsa-miR-423 rs6505162 C>A genotypes were determined using Ligation Detection Reaction (LDR) method. Our results demonstrated that hsa-miR-34b/c rs4938723 CC genotype had a decreased risk of ESCC. The association was evident among patients who never drinking. Hsa-miR-423 rs6505162 C>A might associated with a significantly increased risk of ESCC in patients who smoking. These findings indicated that functional polymorphisms hsa-miR-34b/c rs4938723 T>C and hsa-miR-423 rs6505162 C>A might alter individual susceptibility to ESCC. However, our results were obtained with a limited sample size. Future larger studies with other ethnic populations are required to confirm current findings.     

Blood groups in Native Americans: a look beyond ABO and Rh

The study presents comparisons between blood group frequencies beyond ABO and Rh blood systems in Native American populations and previously published data from Brazilian blood donors. The frequencies of Diego (c.2561C>T, rs2285644), Kell (c.578C>T, rs8176058), Duffy (c.125A>G, rs12075, c.1−67T>C, rs2814778) and Kidd (c.838A>G, rs1058396) variants in Kaingang (n=72) and Guarani (n=234) populations from Brazil (1990-2000) were obtained and compared with data from these populations sampled during the 1960s and with individuals of different Brazilian regions. Data showed high frequencies of DI*01 and FY*01 alleles: 11.8% and 57.6% in Kaingang and 6.8% and 75.7% in Guarani groups, respectively. The main results indicated: (1) reduction in genetic distance over time of Kaingang and Guarani in relation to other Brazilian populations is suggestive of ongoing admixture; (2) significant differences in some frequencies of blood group markers (especially Diego, Kidd and Duffy) in relation to Native Americans and individuals from different geographical regions of Brazil. Our study shows that the frequency of red blood cell polymorphisms in two Native American groups is very different from that of blood donors, when we evaluated blood groups different from ABO and Rh systems, suggesting that a better ethnic characterization of blood unit receptors is necessary.     

Biallelic SUN5 Mutations Cause Autosomal-Recessive Acephalic Spermatozoa Syndrome

Acephalic spermatozoa syndrome is a rare and severe form of teratozoospermia characterized by a predominance of headless spermatozoa in the ejaculate. Family clustering and consanguinity suggest a genetic origin; however, causative mutations have yet to be identified. We performed whole-exome sequencing in two unrelated infertile men and subsequent variant filtering identified one homozygous (c.824C>T [p.Thr275Met]) and one compound heterozygous (c.1006C>T [p.Arg356Cys] and c.485T>A [p.Met162Lys]) SUN5 (also named TSARG4) variants. Sanger sequencing of SUN5 in 15 additional unrelated infertile men revealed four compound heterozygous (c.381delA [p.Val128Serfs^∗7] and c.824C>T [p.Thr275Met]; c.381delA [p.Val128Serfs^∗7] and c.781G>A [p.Val261Met]; c.216G>A [p.Trp72^∗] and c.1043A>T [p.Asn348Ile]; c.425+1G>A/c.1043A>T [p.Asn348Ile]) and two homozygous (c.851C>G [p.Ser284^∗]; c.350G>A [p.Gly114Arg]) variants in six individuals. These 10 SUN5 variants were found in 8 of 17 unrelated men, explaining the genetic defect in 47.06% of the affected individuals in our cohort. These variants were absent in 100 fertile population-matched control individuals. SUN5 variants lead to absent, significantly reduced, or truncated SUN5, and certain variants altered SUN5 distribution in the head-tail junction of the sperm. In summary, these results demonstrate that biallelic SUN5 mutations cause male infertility due to autosomal-recessive acephalic spermatozoa syndrome.     

JAG1 Mutation Spectrum and Origin in Chinese Children with Clinical Features of Alagille Syndrome

Alagille syndrome is an autosomal dominant disorder that results from defects in the Notch signaling pathway, which is most frequently due to JAG1 mutations. This study investigated the rate, spectrum, and origin of JAG1 mutations in 91 Chinese children presenting with at least two clinical features of Alagille syndrome (cholestasis, heart murmur, skeletal abnormalities, ocular abnormalities, characteristic facial features, and renal abnormalities). Direct sequencing and/or multiplex-ligation-dependent probe amplification were performed in these patients, and segregation analysis was performed using samples available from the parents. JAG1 disease-causing mutations were detected in 70/91 (76.9%) patients, including 29/70 (41.4%) small deletions, 6/70 (8.6%) small insertions, 16/70 (22.9%) nonsense mutations, 8/70 (11.4%) splice-site mutations, 6/70 (9.4%) missense mutations, and 5/70 (7.1%) gross deletions. Of the mutations detected, 45/62 (72.6%) were novel, and almost all were unique, with the exception of c.439C>T, c.439+1G>A, c.703C>T, c.1382_1383delAC, c.2698C>T, and c.2990C>A, which were detected in two cases each; three cases exhibited entire gene deletions. A majority (69.2%) of the point and frameshift mutations could be detected by the sequencing of eleven exons (exons 3, 5, 6, 11, 14, 16, 18, 21, and 23–25). The mutation detection rate was 50.0% (10/20) in atypical cases that only presented with two or three clinical features of Alagille syndrome. Segregation analysis revealed that 81.1% (30/37) of these mutations were de novo. In conclusion, JAG1 mutations are present in the majority of Chinese pediatric patients with clinical features of Alagille syndrome, and the mutations concentrate on different exons from other reports. Genetic study is important for the diagnosis of atypical Alagille syndrome in Chinese patients.     

Association of IL10 Polymorphisms and Leprosy: A Meta-Analysis

Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal IL10 polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11–1.34) and P-value = 3x10^–5 confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups (OR = 1.2, CI = 1.10–1.31, P-value = 2x10^–5). Also, meta-analysis confirmed association of -592 A allele and leprosy outcome (OR = 1.24, CI = 1.03–1.50, P-value = 0.02). In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r² = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other IL10 polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.     

DEFB1 polymorphisms are involved in susceptibility to human papillomavirus infection in Brazilian gynaecological patients

The human beta defensin 1 (hBD-1) antimicrobial peptide is a member of the innate immune system known to act in the first line of defence against microorganisms, including viruses such as human papillomavirus (HPV). In this study, five functional polymorphisms (namely g-52G>A, g-44C>G and g-20G>A in the 5’UTR and c.*5G>A and c.*87A>G in the 3’UTR) in the DEFB1 gene encoding for hBD-1 were analysed to investigate the possible involvement of these genetic variants in susceptibility to HPV infection and in the development of HPV-associated lesions in a population of Brazilian women. The DEFB1 g-52G>A and c.*5G>A single-nucleotide polymorphisms (SNPs) and the GCAAA haplotype showed associations with HPV-negative status; in particular, the c.*5G>A SNP was significantly associated after multiple test corrections. These findings suggest a possible role for the constitutively expressed beta defensin-1 peptide as a natural defence against HPV in the genital tract mucosa.     

Riboflavin status modifies the effects of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms on homocysteine

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), riboflavin-dependent enzymes, participate in homocysteine metabolism. Reported effects of riboflavin status on the association between the MTHFR 677C>T polymorphism and homocysteine vary, and the effects of the MTRR 66A>G or MTRR 524C>T polymorphisms on homocysteine are unclear. We tested the hypothesis that the effects of the MTHFR 677C>T, MTRR 66A>G and MTRR 524C>T polymorphisms on fasting plasma total homocysteine (tHcy) depend on riboflavin status (erythrocyte glutathionine reductase activation coefficient, optimum: <1.2; marginally deficient: 1.2–1.4; deficient: ≥1.4) in 771 adults aged 18–75 years. MTHFR 677T allele carriers with middle or low tertile plasma folate (<14.7 nmol/L) had 8.2 % higher tHcy compared to the 677CC genotype (p < 0.01). This effect was eliminated when riboflavin status was optimal (p for interaction: 0.048). In the lowest cobalamin quartile (≤273 pmol/L), riboflavin status modifies the relationship between the MTRR 66 A>G polymorphism and tHcy (p for interaction: 0.034). tHcy was 6.6 % higher in MTRR 66G allele carriers compared to the 66AA genotype with marginally deficient or optimal riboflavin status, but there was no difference when riboflavin status was deficient (p for interaction: 0.059). tHcy was 13.7 % higher in MTRR 524T allele carriers compared to the 524CC genotype when cobalamin status was low (p < 0.01), but no difference was observed when we stratified by riboflavin status. The effect of the MTHFR 677C>T polymorphism on tHcy depends on riboflavin status, that of the MTRR 66A>G polymorphism on cobalamin and riboflavin status and that of the MTRR 524C>T polymorphism on cobalamin status.     

Role of the DLGAP2 Gene Encoding the SAP90/PSD-95-Associated Protein 2 in Schizophrenia

Aberrant synaptic dysfunction is implicated in the pathogenesis of schizophrenia. The DLGAP2 gene encoding the SAP90/PSD-95-associated protein 2 (SAPAP2) located at the post-synaptic density of neuronal cells is involved in the neuronal synaptic function. This study aimed to investigate whether the DLGAP2 gene is associated with schizophrenia. We resequenced the putative promoter region and all the exons of the DLGAP2 gene in 523 patients with schizophrenia and 596 non-psychotic controls from Taiwan and conducted a case-control association analysis. We identified 19 known SNPs in this sample. Association analysis of 9 SNPs with minor allele frequency greater than 5% showed no association with schizophrenia. However, we found a haplotype (CCACCAACT) significantly associated with schizophrenia (odds ratio:2.5, p<0.001). We also detected 16 missense mutations and 1 amino acid-insertion mutation in this sample. Bioinformatic analysis showed some of these mutations were damaging or pathological to the protein function, but we did not find increased burden of these mutations in the patient group. Notably, we identified 5 private rare variants in 5 unrelated patients, respectively, including c.−69+9C>T, c.−69+13C>T, c.−69+47C>T, c.−69+55C>T at intron 1 and c.−32A>G at untranslated exon 2 of the DLGAP2 gene. These rare variants were not detected in 559 control subjects. Further reporter gene assay of these rare variants except c.−69+13C>T showed significantly elevated promoter activity than the wild type, suggesting increased DLGAP2 gene expression may contribute to the pathogenesis of schizophrenia. Our results indicate that DLGAP2 is a susceptible gene of schizophrenia.     

Four Common Vascular Endothelial Growth Factor Polymorphisms (?2578C>A, ?460C>T, +936C>T,and +405G>C) in Susceptibility to Lung Cancer: A Meta-Analysis

Background and Objective

Vascular endothelial growth factor (VEGF) is one of the key initiators and regulators of angiogenesis and it plays a vital role in the onset and development of malignancy. The association between VEGF gene polymorphisms and lung cancer risk has been extensively studied in recent years, but currently available results remain controversial or ambiguous. The aim of this meta-analysis is to investigate the associations between four common VEGF polymorphisms (i.e., −2578C>A, −460C>T, +936C>T and +405C>G) and lung cancer risk.

Methods

A comprehensive search was conducted to identify all eligible studies to estimate the association between VEGF polymorphisms and lung cancer risk. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of this association.

Results

A total of 14 published case-control studies with 4,664 cases and 4,571 control subjects were identified. Our meta-analysis provides strong evidence that VEGF −2578C>A polymorphism is capable of increasing lung cancer susceptibility, especially among smokers and lung squamous cell carcinoma (SCC) patients. Additionally, for +936C>T polymorphism, increased lung cancer susceptibility was only observed among lung adenocarcinoma patients. In contrast, VEGF −460C>T polymorphism may be a protective factor among nonsmokers and SCC patients. Nevertheless, we did not find any association between +405C>G polymorphism and lung cancer risk, even when the groups were stratified by ethnicity, smoking status or histological type.

Conclusion

This meta-analysis recommends more investigations into the relationship between −2578C>A and −460C>T lung cancer risks. More detailed and well-designed studies should be conducted to identify the causal variants and the underlying mechanisms of the possible associations.     

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose

Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.

Methods

Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.

Results

Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).

Conclusions

Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.     

MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8⁺ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations.     

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.     

TERT-CLPTM1L Rs401681 C>T Polymorphism Was Associated with a Decreased Risk of Esophageal Cancer in a Chinese Population

Background

Esophageal cancer was the fifth most commonly diagnosed cancer and the fourth leading cause of cancer-related death in China in 2009. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90 percent of esophageal cancers. Genetic factors probably play an important role in the ESCC carcinogenesis.

Methods

We conducted a hospital based case-control study to evaluate functional hTERT rs2736098 G>A and TERT-CLPTM1L rs401681 C>T single nucleotide polymorphisms (SNPs) on the risk of ESCC. Six hundred and twenty-nine ESCC cases and 686 controls were recruited. Their genotypes were determined using the ligation detection reaction (LDR) method.

Results

When the TERT-CLPTM1L rs401681 CC homozygote genotype was used as the reference group, the CT genotype was associated with a significantly decreased risk of ESCC (adjusted OR  = 0.74, 95% CI  = 0.58–0.94, p = 0.012); the CT/TT variants were associated with a 26% decreased risk of ESCC (adjusted OR  = 0.74, 95% CI  = 0.59–0.93, P = 0.009). The significantly decreased risk of ESCC associated with the TERT-CLPTM1L rs401681 C>T polymorphism was associated with male sex, young age (<63 years in our study) and alcohol consumption. No association between the hTERT rs2736098 G>A polymorphism and ESCC risk was observed.

Conclusion

TERT-CLPTM1L rs401681 CT and CT/TT genotypes were associated with decreased risk of ESCC, particularly among men, young patients and those reported to be drinkers. However, our results are preliminary conclusions. Larger studies with more rigorous study designs are required to confirm the current findings.     

A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1

Retinoblastoma (Rb), the most common pediatric intraocular neoplasm, results from inactivation of both alleles of the RB1 tumor suppressor gene. The second allele is most commonly lost, as demonstrated by loss of heterozygosity studies. RB1 germline carriers usually develop bilateral tumors, but some Rb families display low penetrance and variable expressivity. In order to decipher the underlying mechanisms, 23 unrelated low penetrance pedigrees segregating the common c.1981C>T/p.Arg661Trp mutation and other low penetrance mutations were studied. In families segregating the c.1981C>T mutation, we demonstrated, for the first time, a correlation between the gender of the transmitting carrier and penetrance, as evidenced by Fisher’s exact test: the probability of being unaffected is 90.3% and 32.5% when the mutation is inherited from the mother and the father, respectively (p-value = 7.10⁻⁷). Interestingly, a similar correlation was observed in families segregating other low penetrance alleles. Consequently, we investigated the putative involvement of an imprinted, modifier gene in low penetrance Rb. We first ruled out a MED4-driven mechanism by MED4 methylation and expression analyses. We then focused on the differentially methylated CpG85 island located in intron 2 of RB1 and showing parent-of-origin-specific DNA methylation. This differential methylation promotes expression of the maternal c.1981C>T allele. We propose that the maternally inherited c.1981C>T/p.Arg661Trp allele retains sufficient tumor suppressor activity to prevent retinoblastoma development. In contrast, when the mutation is paternally transmitted, the low residual activity would mimic a null mutation and subsequently lead to retinoblastoma. This implies that the c.1981C>T mutation is not deleterious per se but needs to be destabilized in order to reach pRb haploinsufficiency and initiate tumorigenesis. We suggest that this phenomenon might be a general mechanism to explain phenotypic differences in low penetrance Rb families.     

Correlations of MTHFR 677C>T Polymorphism with Cardiovascular Disease in Patients with End-Stage Renal Disease: A Meta-Analysis

ObjectiveThis meta-analysis was conducted to evaluate the correlations of a common polymorphism (677C>T) in the methylenetetrahydrofolate reductase (MTHFR) gene with risk of cardiovascular disease (CVD) in patients with end-stage renal disease (ESRD).MethodThe following electronic databases were searched without language restrictions: Web of Science (1945∼2013), the Cochrane Library Database (Issue 12, 2013), MEDLINE (1966∼2013), EMBASE (1980∼2013), CINAHL (1982∼2013) and the Chinese Biomedical Database (CBM) (1982∼2013). Meta-analysis was performed using STATA statistical software. Odds ratios (ORs) with their 95% confidence intervals (95%CIs) were calculated.ResultsEight cohort studies met all inclusion criteria and were included in this meta-analysis. A total of 2,292 ESRD patients with CVD were involved in this meta-analysis. Our meta-analysis results revealed that the MTHFR 677C>T polymorphism might increase the risk of CVD in ESRD patients (TT vs. CC: OR = 2.75, 95%CI = 1.35∼5.59, P = 0.005; CT+TT vs. CC: OR = 1.39, 95%CI = 1.09∼1.78, P = 0.008; TT vs. CC+CT: OR = 2.52, 95%CI = 1.25∼5.09, P = 0.010; respectively). Further subgroup analysis by ethnicity suggested that the MTHFR 677C>T polymorphism was associated with an elevated risk for CVD in ESRD patients among Asians (TT vs. CC: OR = 3.38, 95%CI = 1.11∼10.28, P = 0.032; CT+TT vs. CC: OR = 1.44, 95%CI = 1.05∼1.97, P = 0.022; TT vs. CC+CT: OR = 3.15, 95%CI = 1.02∼9.72, P = 0.046; respectively), but not among Africans or Caucasians (all P>0.05).ConclusionOur findings indicate that the MTHFR 677C>T polymorphism may be associated with an elevated risk for CVD in ESRD patients, especially among Asians.     

Multiplex Amplicon Genotyping by High-Resolution Melting

High-resolution amplicon melting is a simple method for genotyping that uses only generic PCR primers and a saturating DNA dye. Multiplex amplicon genotyping has previously been reported in a single color, but two instruments were required: a carousel-based rapid cycler and a high-resolution melting instrument for capillaries. Manual transfer of capillaries between instruments and sequential melting of each capillary at 0.1°C/s seriously limited the throughput. In this report, a single instrument that combines rapid-cycle real-time PCR with high-resolution melting [LightScanner-32 (LS-32), Idaho Technology, Salt Lake City, UT] was used for multiplex amplicon genotyping. The four most common mutations associated with thrombophilia, F5 (factor V Leiden 1691G>A), F2 (prothrombin 20210G>A), and methylenetetrahydrofolate reductase (MTHFR; 1298A>C and 677C>T) were genotyped in a single homogeneous assay with internal controls to adjust for minor chemistry and instrument variation. Forty temperature cycles required 9.2 min, and each capillary required 2.2 min by melting at 0.3°C/s, 3× the prior rate. Sample volume was reduced from 20 μl to 10 μl. In a blinded study of 109 samples (436 genotypes), complete concordance with standard assays was obtained. In addition, the rare variant MTHFR 1317T>C was genotyped correctly when present. The LS-32 simplifies more complex high-resolution melting assays by reducing hands-on manipulation, total time of analysis, and reagent cost while maintaining the resolution necessary for multiplex amplicon genotyping.     

KCNJ10 May Not Be a Contributor to Nonsyndromic Enlargement of Vestibular Aqueduct (NSEVA) in Chinese Subjects

Background

Nonsyndromic enlargement of vestibular aqueduct (NSEVA) is an autosomal recessive hearing loss disorder that is associated with mutations in SLC26A4. However, not all patients with NSEVA carry biallelic mutations in SLC26A4. A recent study proposed that single mutations in both SLC26A4 and KCNJ10 lead to digenic NSEVA. We examined whether KCNJ10 excert a role in the pathogenesis of NSEVA in Chinese patients.

Methods

SLC26A4 was sequenced in 1056 Chinese patients with NSEVA. KCNJ10 was screened in 131 patients who lacked mutations in either one or both alleles of SLC26A4. Additionally, KCNJ10 was screened in 840 controls, including 563 patients diagnosed with NSEVA who carried biallelic SLC26A4 mutations, 48 patients with nonsyndromic hearing loss due to inner ear malformations that did not involve enlargement of the vestibular aqueduct (EVA), 96 patients with conductive hearing loss due to various causes, and 133 normal-hearing individuals with no family history of hereditary hearing loss.

Results

925 NSEVA patients were found carrying two-allele pathogenic SLC26A4 mutations. The most frequently detected KCNJ10 mutation was c.812G>A (p.R271H). Compared with the normal-hearing control subjects, the occurrence rate of c.812G>A in NSEVA patients with lacking mutations in one or both alleles of SLC26A4 had no significant difference(1.53% vs. 5.30%, χ² = 2.798, p = 0.172), which suggested that it is probably a nonpathogenic benign variant. KCNJ10 c.1042C>T (p.R348C), the reported EVA-related mutation, was not found in patients with NSEVA who lacked mutations in either one or both alleles of SLC26A4. Furthermore, the normal-hearing parents of patients with NSEVA having two SLC26A4 mutations carried the KCNJ10 c.1042C>T or c.812G>A mutation and a SLC26A4 pathogenic mutation.

Conclusion

SLC26A4 is the major genetic cause in Chinese NSEVA patients, accounting for 87.59%. KCNJ10 may not be a contributor to NSEVA in Chinese population. Other genetic or environmental factors are possibly play a role in the etiology of Chinese EVA patients with zero or monoallelic SLC26A4 mutation.