Sequencing Of Coding Exons Research Articles

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

Variation in the ovine FOXP3 gene and its effect on growth traits in Egyptian Barki sheep

The aim of the present study was to detect the FOXP3 gene polymorphisms in Barki sheep at a variable region covering exon 13, intron 13 and the coding sequence in exon 14 and to test the association of these polymorphisms with growth traits. 122 Barki lambs were phenotyped for various growth traits, viz., birth weight (BW), weaning weight (WW), pre-weaning daily gain in weight (ADG1), post-weaning daily gain in weight (ADG2) and marketing bodyweight (MW). The polymerase chain reaction – single-strand conformational polymorphisms (PCR-SSCP) and DNA sequencing methods were used to identify the genetic variants in the FOXP3 gene. The associations between the variation in FOXP3 gene and growth traits were tested using a general linear model. Two variants (F1 and F2 with gene frequencies of 0.64 and 0.36, respectively), and three genotypes (F1F1, F1F2 and F2F2 with frequencies of 0.37, 0.53 and 0.10, respectively) were detected. The association of FOXP3 genotype was significant (p < 0.05) with ADG2 and MW. It is concluded that FOXP3 genotype might be helpful for sheep breeders to produce fast-growing lambs. However, further studies are needed in a large population to confirm the association we found.

CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A.

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2–26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.

Specific and Sensitive Diagnosis of BCOR-ITD in Various Cancers by Digital PCR.

BCOR is an epigenetic regulator altered by various mechanisms including BCOR-internal tandem duplication (BCOR-ITD) in a wide range of cancers. Six different BCOR-ITD in the 3’-part of the coding sequence of exon 15 have been reported ranging from 89 to 114 bp in length. BCOR-ITD is a common genetic alteration found in clear cell sarcoma of the kidney and primitive myxoid mesenchymal tumor of infancy (PMMTI) and it characterizes a new type of central nervous system tumor: “CNS tumor with BCOR-ITD”. It can also be detected in undifferentiated round cell sarcoma (URCS) and in high-grade endometrial stromal sarcoma (HGESS). Therefore, it is of utmost importance to search for this genetic alteration in these cancers with the most frequent technique being RNA-sequencing. Here, we developed a new droplet PCR assay (dPCR) to detect the six sequences characterizing BCOR-ITD. To achieve this goal, we used a single colored probe to detect both the duplicated region and the normal sequence that acts as a reference. We first generated seven synthetic DNA sequences: ITD0 (the normal sequence) and ITD1 to ITD6 (the duplicated sequences described in the literature) and then we set up the optima dPCR conditions. We validated our assay on 19 samples from a representative panel of human tumors (9 HGNET-BCOR, 5 URCS, 3 HGESS, and 2 PMMTI) in which BCOR-ITD status was known using at least one other method including RNA sequencing, RT-PCR or DNA-methylation profiling for CNS tumors. Our results showed that our technique was 100% sensitive and specific. DPCR detected BCOR-ITD in 13/19 of the cases; in the remaining 6 cases additional RNA-sequencing revealed BCOR gene fusions. To conclude, in the era of histomolecular classification of human tumors, our modified dPCR assay is of particular interest to detect BCOR-ITD since it is a robust and less expensive test that can be applied to a broad spectrum of cancers that share this alteration.

Identification of Novel Missense Mutations of the TSH-β Subunit Gene in Pediatric Patients with Congenital Central Hypothyroidism

Background: Congenital central hypothyroidism (CCH) is a rare autosomal recessive disease caused by mutations in the thyroid-stimulating hormone β subunit (TSHβ) gene. Since patients with CCH do not experience increased serum levels of TSH, the diagnosis is usually delayed, which leads to negative consequences in the neonatal TSH screening. Genetic diagnostic studies enable us to identify affected relatives at high risk for rapid diagnosis and treatment of the disorder. Objectives: This study aimed to investigate genetic variations in the TSHβ gene for the first time in Iranian patients with CCH. Methods: Seven children affected by congenital TSH-deficient hypothyroidism were investigated for mutations in TSHβ. Variable TSH levels in these patients ranged from low values for diagnosis to significant values, so central hypothyroidism was assumed due to mutations in the TSHβ gene. Results: We identified two novel heterozygous (F11Y and G106R) and one homozygous (T14A) missense mutations in the coding sequence of exons 2 and 3. One of the new heterozygous mutations (F11Y) and a homozygous (T14A) missense mutation were found in exon 2 of the TSHß-subunit gene. The novel mutation G106R in exon 3 was found in three pediatric patients with congenital hypothyroidism. c.40A&gt;G (T14A, rs10776792) appears to be the most common genetic variation associated with TSH deficiency. The others were c.32T&gt;A in exon 2 and c.316G&gt;C in exon 3, which resulted in a change from phenylalanine to tyrosine (p.F11Y) and glycine to arginine (G106R), respectively. Conclusions: The identification of these mutations for the first time in Iranian patients suggests that CCH is more common than previously recognized, and the TSHβ gene may be the mutational hot spot.

Short Communication: Identification of Leptin gene in crossbred beef cattle

Abstract. Anugratama LE, Hartatik T. 2020. Short Communication: Identification of Leptin gene in crossbred beef cattle. Biodiversitas 21: 226-230. Leptin is a gene that affects animal weight. Leptin gene is known to control body weight, feed intake, energy expenditure, immune function, and reproduction. This study aims to identify the diversity of the Leptin gene in crossbred beef cattle, Sumba Ongole cattle, Brahman cross cattle, Bali cattle, buffalo, sheep, and goat by comparing with four GenBank data of cattle. Crossbred beef cattle obtained from Klaten, Central Java, Indonesia. Leptin nucleotide sequences were analyzed using BioEdit to identify Single Nucleotide Polymorphism (SNP). To create amino acid change in Leptin gene, the coding sequence of exon 2 was established using BioEdit ver. 7.0.5. Phylogenetic tree and genetic distance have been analyzed based on the Leptin gene using MEGA 10.1.1 program. The result shows that eight variations of SNP were found in exon 2. The phylogenetic tree represents that crossbreed beef cattle, Sumba Ongole cattle, Brahman cross cattle, Bali cattle, Bos taurus, Bos indicus, Bos frontalis, Bos grunniens, Bubalus bubalis are in the same cluster with various genetic distance. The results of this study are expected to provide genetic information that will be used for further research on the relationship between Leptin gene polymorphisms to animal weight.

A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in CNS disorders is not clear due to lack of tools to discriminate between microglia and infiltrating myeloid cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker, TMEM119, for studying microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3′UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 in the CNS of Tmem119tdTomato/+ relative to wild-type mice. The cells expressing tdTomato were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Additionally, immunofluorescence and flow cytometry techniques revealed that tdTomato+ cells are detected throughout the CNS, but not in peripheral tissues of Tmem119tdTomato/+ mice. Aging does not influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that tdTomato+ cells colocalize with Iba1+ cells in the brain, but not with neurons, astrocytes or oligodendrocytes. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglia CD45loCD11b+ cells (96.3%) are tdTomato-positive; and a minority of infiltrating CD45hiCD11b+ myeloid cells (5.3%) are also tdTomato-positive, which we further characterized and found that tdTomato expression is in part of choroid plexus macrophages but not in meningeal and perivascular macrophages. Functionally, using an acute injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model is a valuable tool to specifically study the role of microglia in health and disease.

Rapid identification of the purple stem (Ps) gene of Chinese kale (Brassica oleracea var. alboglabra) in a segregation distortion population by bulked segregant analysis and RNA sequencing

Abundant anthocyanins accumulate in many flowers, leaves, fruits, as well as stems. In this study, an F2 segregating population derived from a cross between Chinese kale with a purple stem and kale with a green stem was used to decipher the genetic basis for the stem color. Bulked segregant analysis in combination with RNA sequencing (BSR-seq) was used to genetically map the causal gene for the purple stem (Ps), and the allele frequency difference between the two pools showed a single peak on chromosome C09. Ps was fine-mapped to the 0.32 cM interval with 406 kb sequences. A gene-encoding dihydroflavonol 4-reductase (DFR), an enzyme in the anthocyanin biosynthesis pathway, was differentially expressed between the two pools, and was identified as the candidate gene for the purple stem in Chinese kale. Sequence analysis revealed that 1 bp was inserted in the coding sequence of exon 2 of the BoDRF gene in green kale, causing frameshift and loss of function. The expression level of the BoDFR gene was significantly lower than the Chinese kale cultivar with a purple stem. The genetic results of the Ps gene will be useful for future development of purple vegetables.

Recent evolution of the salivary mucin MUC7.

Genomic structural variants constitute the majority of variable base pairs in primate genomes and affect gene function in multiple ways. While whole gene duplications and deletions are relatively well-studied, the biology of subexonic (i.e., within coding exon sequences), copy number variation remains elusive. The salivary MUC7 gene provides an opportunity for studying such variation, as it harbors copy number variable subexonic repeat sequences that encode for densely O-glycosylated domains (PTS-repeats) with microbe-binding properties. To understand the evolution of this gene, we analyzed mammalian and primate genomes within a comparative framework. Our analyses revealed that (i) MUC7 has emerged in the placental mammal ancestor and rapidly gained multiple sites for O-glycosylation; (ii) MUC7 has retained its extracellular activity in saliva in placental mammals; (iii) the anti-fungal domain of the protein was remodified under positive selection in the primate lineage; and (iv) MUC7 PTS-repeats have evolved recurrently and under adaptive constraints. Our results establish MUC7 as a major player in salivary adaptation, likely as a response to diverse pathogenic exposure in primates. On a broader scale, our study highlights variable subexonic repeats as a primary source for modular evolutionary innovation that lead to rapid functional adaptation.

A summary of molecular genetic findings in fructose-1,6-bisphosphatase deficiency with a focus on a common long-range deletion and the role of MLPA analysis.

BackgroundFructose-1,6-bisphosphatase deficiency is a rare inborn error of metabolism affecting gluconeogenesis with only sporadic reports on its molecular genetic basis.ResultsWe report our experience with mutation analysis in 14 patients (13 families) with fructose-1,6-bisphosphatase deficiency using conventional Sanger sequencing and multiplex ligation-dependent probe amplification analysis, and we provide a mutation update for the fructose bisphosphatase-1 gene (FBP1). Mutations were found on both chromosomes in all of our 14 patients including 5 novel mutations. Among the novel mutations is a 5412-bp deletion (c.-24-26_170 + 5192del) including the entire coding sequence of exon 2 of FBP1 that was repeatedly found in patients from Turkey and Armenia which may explain earlier poorly defined findings in patients from this area. This deletion can be detected with specific primers by generation of a junction fragment and by MLPA and SNP array assays. MLPA analysis was able to detect copy number variations in two further patients, one heterozygous for a deletion within exon 8, another heterozygous for a novel deletion of the entire FBP1 gene.ConclusionsBased on our update for the FBP1 gene, currently listing 35 mutations worldwide, and knowledge of PCR conditions that allow simple detection of a common FBP1 deletion in the Armenian and Turkish population, molecular genetic diagnosis has become easier in FBP1 deficiency. Furthermore, MLPA analysis may plays a useful role in patients with this disorder.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0415-1) contains supplementary material, which is available to authorized users.

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

Сравнительный анализ мутаций в гене ТР53 у больных ДВККЛ г.Новосибирска с данными, представленными в IARC TP53 mutation database

Relevance. The information about the frequency and spectrum of TP53 gene mutations in Russian patients with diffuse large cell lymphoma (DLBCL) is not represented in the current version of the IARC TP53 mutation database. Purpose of the study was to compare the frequency and spectrum of TP53 gene mutations in Novosibirsk patients with DLBCL with the data presented in IARC TP53 mutation database. Material and methods. The TP53 gene sequence from 5 to 10 exons of 74 tumor tissue samples of patients with newly diagnosed DLBCL was analyzed by Sanger direct sequencing. Results. In 24.3% of patients were identified a mutation in the coding sequence of exons 5-8 TP53 gene. Multiple mutations had 4% of patients. The cases of loss of heterozygosity in the TP53 gene in DLBCL tumor tissue were revealed. The spectrum of single-nucleotide substitutions in the TP53 in the study group did not significantly differ from the data presented in IARC TP53 mutation database, but we observed differences in localization of the «hot spot» mutations. In the analyzed group of patients with DLBCL «hot spots» mutations were in codons 275, 155, 272 and 212. Conclusions. Comparative analysis of the results of sequencing gene TP53 in tumor tissue of patients with DLBCL in Novosibirsk with the data presented in IARC TP53 mutation database is revealed differences in the spectrum of mutations. The frequency of mutations in the gene TP53 in the study group are consistent with the literature dates.

Relationship between mutation of BRCA1 and susceptibility to early onset of breast cancer

To detect the prevalence of Breast Cancer Susceptibility Gene 1 (BRCA1) mutations and single nucleotide polymorphism (SNP) among young patients with breast cancer and to study the relationship between BRCA1 gene mutation and susceptibility to breast cancer. 30 samples of breast cancer tissue were collected from female patients with breast cancer diagnosed when they were aged < or = 35 5 of which had at least one first-degree relative affected with breast cancer. Genomic DNA was extracted from the breast cancer tissues. The PCR products were amplified in the coding sequence of exon 2, 11C, 11F, 11L, 11I, 16, and 20 by using polymerase chain reaction. Then the PCR products were analyzed using DNA direct sequencing. The sequence was compared with the DNA Star-MagAlign software. A total of 14 sequence variations in BRCA1 gene were identified, including 3 frameshift mutations (cDNA2639, 2640delTA, 3343 delG, and 3398delT) and 11 spot mutations (cDNA 2570 C > T, cDNA 2620 A > T, 1473 A > G, 1561 C > T, 1594G > A, 2206 A > G, 2227 T > C, 2659 C > A, 2806T > C, 3307 A > G, and 3375 G > A). Three patients with these mutations had a family history of breast cancer. The mutation frequency of BRCA1 was 10% (3/30). No mutation was found in the exons 16 and 20. Patients with breast cancer aged < or = 35 have BRCA1 mutations located in the exon 11 mainly. The mutation frequency of the breast cancer patients aged < or = 35 with breast cancer family history is higher than those without family history. Three frameshift mutation sites may be related to early onset of breast cancer.

Genetic variations in the GLUT3 gene associated with myelomeningocele

Our objectives were to examine the extent of described sequence variation in the glucose transporter 3 (GLUT3) gene in children with myelomeningocele (MM), identify novel variations in the GLUT3 gene in these children, and determine whether these variations may confer a risk of MM. We sequenced the 10 exons of GLUT3, including exon-intron boundaries, on 96 children with MM. Sequencing was performed with Sanger methods and results analyzed with deoxyribonucleic acid analysis software. Frequencies of known single-nucleotide polymorphisms were identified, and those differing from the reference sequence (GRCh37/hg19 assembly) were considered variations. Six novel and 9 previously described, genetic variations were identified in our population. The novel variations included a large, 83 base pair deletion involving the core promoter region and part of exon 1 (1 of 96 children), and a 2 base pair deletion in the coding sequence of exon 4 (1 of 96 children). The remaining novel variations were located in the introns in the proximity of the splice sites. Novel mutations in GLUT3 were observed among 6.25% of our population. Additionally, the frequency of the rare allele for rs17847972 located in a splice donor site is higher (P < .001) in MM in our population than expected. We identified previously undescribed deletions and single-nucleotide variations involving the GLUT3 gene that may be associated with increased susceptibility to MM. Of particular interest, the 2 deletions involve both an important core promoter site and a coding region predicted to have a deleterious effect. The functional significance of these findings is under investigation.

TaGS-D1, an ortholog of rice OsGS3, is associated with grain weight and grain length in common wheat

The OsGS3 gene plays a principal role in controlling grain weight and grain length in rice. However, the function of an orthologous gene TaGS in wheat has not been analyzed to date. In the present study, we cloned the gDNA of TaGS gene, designated TaGS-D1, with four exons and three introns on chromosome 7DS by a comparative genomics approach. The cDNA of TaGS-D1 is 255 bp, and it encodes 85 amino acids. We also found a plant-specific organ size regulation domain in the deduced polypeptide, indicating that TaGS-D1, like OsGS3, does not belong to the PEBP family. DNA sequencing of the TaGS-D1 locus revealed no diversity in the coding sequence of exons, but there was a single nucleotide polymorphism (SNP) in the first intron, and 30 SNPs, a 40-bp InDel and a 3-bp InDel were found in the second intron between genotypes with higher and lower thousand grain weights (TGW). Based on the 40-bp InDel, a co-dominant STS marker, designated GS7D, was developed to discriminate the two alleles. GS7D was 8.0 cM from Xbarc184 located on chromosome 7DS by linkage mapping. A QTL for TGW and grain length at GS7D locus explained up to 16.3 and 7.7 %, respectively, of the phenotypic variances in a RIL population derived from Doumai/Shi 4185 grown in Shijiazhuang and Beijing. One hundred and seventy-five Chinese wheat cultivars were genotyped with GS7D, indicating that TaGS-D1 was significantly associated with grain weight. The allelic distribution at the TaGS-D1 locus showed that the frequencies of TaGS-D1a were high in cultivars from Serbia, Japan, Australia, Canada, and the Northeastern Spring Wheat and Northern Winter Wheat Regions of China.

Sodium/proton exchanger 3 (NHE3) and sudden infant death syndrome (SIDS)

The sodium/proton exchanger protein 3 (NHE3) is located in chemosensitive areas of the medulla oblongata and plays an important role in the central control of respiration. Overexpression of NHE3 is correlated with lower respiration and might therefore contribute to the vulnerability of infants dying suddenly and unexpected (sudden infant death syndrome, SIDS). Our aim in this study was to verify already reported genetic variations in the NHE3 gene in an independent SIDS cohort from Switzerland. Two single nucleotide polymorphisms (SNPs) in the promoter region (G1131A and C1197T) and one variation in the coding sequence of exon 16 (C2405T) in the NHE3 gene were analyzed in 160 Caucasian SIDS infants and 192 Swiss adult controls by using a single base extension method (SNaPshot multiplex). No significant differences were detected in the allelic frequencies of the three NHE3 polymorphisms between SIDS cases and controls. We conclude that the three investigated NHE3 SNPs are unlikely to play a major role in the pathogenesis of SIDS in Caucasian infants. However, further genetic investigations in different ethnicities are required to determine whether variations in NHE3 are associated with an increased SIDS risk.

Single Molecule Characterization of Pre-mRNA Dynamics throughout Spliceosome Assembly and Catalysis

Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Genetic and biochemical techniques have been the preferred tools utilized to identify and characterize splicing complexes throughout spliceosome assembly and catalysis. Unfortunately, these techniques fail to capture the inherent dynamic and heterogeneous nature of the process, a characteristic perhaps most exemplified by the fact that both chemical steps of splicing are reversible processes. In addition, these techniques often focus on the role of spliceosomal components and fail to capture the important information gained by directly observing the conformation of the pre-mRNA at the core of the spliceosome.In an effort to address these limitations, single molecule fluorescence microscopy has recently emerged as an alternative approach. Using a dual-labeled, fluorescent substrate we are able to observe time and ATP-dependent conformational rearrangements of single pre-mRNA molecules throughout spliceosome assembly and catalysis. In addition, substrate and extract mutations allow us to stall assembly at specific stages and enrich for the conformational behaviors characteristic to that stage of splicing. To help us understand the complex behaviors of thousands of molecules interconverting between multiple states with a variety of kinetic behaviors, we have developed a clustering analysis tool that allows us to first classify these diverse behaviors and then assign dynamics to specific complexes along the splicing pathway. This approach is vital to the single molecule field because it allows for an objective characterization of complex systems involving more than two or three states.

G0S2–A new player in leukemia

G0S2–A new player in leukemia

Clinical and genetic analysis in a patient with primary renal glucosuria: Identification of a novel mutation in the SLC5A2 gene

Primary renal glucosuria (PRG; OMIM #233100) is characterized by persistent glucosuria due to a reduction in the renal tubular reabsorption of glucose in the presence of a normal concentration of serum glucose and the absence of any other impairment of tubular function. The SLC5A2 gene is the causative gene, which codes for the low-affinity sodium/glucose co-transporter SGLT2. In the present study, the case of a patient with PRG associated with a novel mutation of the SLC5A2 gene is reported. The patient visited hospital for the evaluation of glucosuria in the absence of hyperglycemia, a condition that had been present for >20 years. The patient showed a fasting blood sugar level of 104 mg/dl, a 2-h postprandial sugar level of 101 mg/dl, a sodium level of 144 mmol/l, a potassium level of 3.7 mmol/l and a chloride level of 106 mmol/l in serum. Urine chemistry revealed that the amount of glucose excreted was 10.8 g/1.73 m2/24 h; however, the levels of the other parameters were unremarkable. Polymerase chain reaction (PCR) sequencing analysis of the SLC5A2 gene from the patient revealed a novel 1 bp deletion mutation, which altered the coding sequence of exon 10 in the transmembrane domain (c.1162delG; Ala388ProfsX48), suggesting an autosomal dominant inheritance pattern. This study identified a novel mutation in the SLC5A2 gene related to a benign clinical characteristic and suggests that the molecular diagnosis of the SLC5A2 gene may be useful for diagnosing renal glucosuria in patients and for deciding intervention measures for their family members.

Resequencing the Whole MYH7 Gene (Including the Intronic, Promoter, and 3′ UTR Sequences) in Hypertrophic Cardiomyopathy

MYH7 mutations are found in ~20% of hypertrophic cardiomyopathy (HCM) patients. Currently, mutational analysis is based on the sequencing of the coding exons and a few exon-flanking intronic nucleotides, resulting in omission of single-exon deletions and mutations in internal intronic, promoter, and 3' UTR regions. We amplified and sequenced large MYH7 fragments in 60 HCM patients without previously identified sarcomere mutations. Lack of aberrant PCR fragments excluded single-exon deletions in the patients. Instead, we identified several new rare intronic variants. An intron 26 single nucleotide insertion (-5 insC) was predicted to affect pre-mRNA splicing, but allele frequencies did not differ between patients and controls (n = 150). We found several rare promoter variants in the patients compared to controls, some of which were in binding sites for transcription factors and could thus affect gene expression. Only one rare 3' UTR variant (c.*29T>C) found in the patients was absent among the controls. This nucleotide change would not affect the binding of known microRNAs. Therefore, MYH7 mutations outside the coding exon sequences would be rarely found among HCM patients. However, changes in the promoter region could be linked to the risk of developing HCM. Further research to define the functional effect of these variants on gene expression is necessary to confirm the role of the MYH7 promoter in cardiac hypertrophy.