SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs

UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which we show here are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with our finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. We discuss the remarkable balance between SMD and the two types of NMD in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis.     

Examination of PPP1R3B as a candidate gene for the type 2 diabetes and MODY loci on chromosome 8p23

The product of the PPP1R3B gene (G_L) is the regulatory subunit of PP1 ‐ a serine/threonine phosphatase involved in the modulation of glycogen synthesis in the liver and skeletal muscle. The PPP1R3B gene is located on chromosome 8p23 in a region that has been linked with type 2 diabetes and maturity‐onset diabetes of the young (MODY). We examined whether sequence variants at the PPP1R3B locus are responsible for the linkage with diabetes observed at this location. RT‐PCR analysis revealed the existence of two alternative promoters. These and the two exons of this gene were sequenced in the probands of 13 Joslin families showing the strongest evidence of linkage at 8p23. A total of 20 variants were observed: two in the 5′ flanking region, one in the intron (9 bp 5′ of exon 2), and 17 in the 3′ UTR. The intronic variant generated a new acceptor splice site, resulting in an alternative splice variant with a longer 5′ UTR. However, neither this nor other variants segregated with diabetes in the 13 ‘linked’ families. Furthermore, allele frequencies were similar in 90 family probands from the Joslin Study and 347 unrelated controls. Thus, genetic variability in the PPP1R3B gene does not appear to contribute to diabetes in our mostly Caucasian families. However, a role cannot be excluded in other populations such as the Japanese, among whom linkage to diabetes is also observed at 8p23 and a non‐synonymous mutation has been detected in the PPP1R3B gene.     

一个先天性小眼畸形家系的全外显子组测序分析及产前诊断

目的分析1个先天性小眼畸形家系的临床表型及遗传学病因。方法应用高通量测序技术对先证者及其父母进行全外显子组测序,筛选候选致病位点,对其家系进行Sanger测序验证,并通过羊水穿刺和Sanger测序为先证者母亲提供产前诊断。结果全外显子组测序和Sanger测序发现家系中的3例患者均携带OTX2基因c.289C>T(p.R97*)杂合变异,先证者母亲亦携带该变异,但无小眼畸形。先证者的父亲、舅母和胎儿未携带上述变异。结论OTX2基因c.289C>T(p.R97*)杂合变异很可能是该家系的发病原因。上述诊断将有助于该家系的遗传咨询和产前诊断。     

Highly recurrent H3F3A mutations with additional epigenetic regulator alterations in giant cell tumor of bone

Recurrent H3F3A and IDH2 mutations have been reported in giant cell tumor of bone (GCTB). However, the reported incidences have varied, and other molecular genetic alterations have not been identified due to the small number of cases analyzed with comprehensive methods. Moreover, the relative sensitivities of Sanger sequencing and next‐generation sequencing (NGS) for the detection of H3F3A mutations in DNA extracted from archival formalin‐fixed paraffin‐embedded (FFPE) samples for clinical diagnosis have not been assessed. To address these issues, we conducted whole‐exome sequencing of 7 GCTBs and integrated the previously published genomic sequencing data of 6 GCTBs. We subsequently performed targeted sequencing of an additional 39 GCTBs, including 2 atypical cases and an extremely rare case of primary malignant transformation of GCTB. We also evaluated the sensitivity of Sanger sequencing for detecting H3F3A mutations in FFPE samples that are usually used for clinical diagnosis. H3F3A glycine hotspot mutations were the most frequently detected mutations (96%) in the 52 GCTBs by NGS. Of the 50 hotspot mutations, p.G34W was observed in 48 cases and p.G34L/G34R was detected in one. One of two atypical GCTB cases with wild‐type H3F3A had a H3F3B mutation (p.G34V). Other mutated genes were not recurrent. Sanger sequencing did not detect H3F3A mutations in 10 of 15 H3F3A NGS mutation‐positive FFPE samples. In conclusion, we confirmed that H3F3A is the most frequently mutated GCTB driver gene, and that H3F3A mutations are not present in atypical GCTBs. Sanger sequencing was much less sensitive than targeted NGS for detecting H3F3A mutations in FFPE samples.     

Identification of pathogenic variants of ERLEC1 in individuals with Class III malocclusion by exome sequencing

Class III malocclusion is a common dentofacial deformity. The underlying genetic alteration is largely unclear. In this study, we sought to determine the genetic etiology for Class III malocclusion. A four‐generation pedigree of Class III malocclusion was recruited for exome sequencing analyses. The likely causative gene was verified via Sanger sequencing in an additional 90 unrelated sporadic Class III malocclusion patients. We identified a rare heterozygous variant in endoplasmic reticulum lectin 1 (ERLEC1; NM_015701.4(ERLEC1_v001):c.1237C>T, p.(His413Tyr), designated as ERLEC1‐m in this article) that cosegregated with the deformity in pedigree members and three additional rare missense heterozygous variants (c.419C>G, p.(Thr140Ser), c.419C>T, p.(Thr140Ile), and c.1448A>G, p.(Asn483Ser)) in 3 of 90 unrelated sporadic subjects. Our results showed that ERLEC1 is highly expressed in mouse jaw osteoblasts and inhibits osteoblast proliferation. ERLEC1‐m significantly enhanced this inhibitory effect of osteoblast proliferation. Our results also showed that the proper level of ERLEC1 expression is crucial for proper osteogenic differentiation. The ERLEC1 variant identified in this study is likely a causal mutation of Class III malocclusion. Our study reveals the genetic basis of Class III malocclusion and provides insights into the novel target for clinical management of Class III malocclusion, in addition to orthodontic treatment and orthodontic surgery.     

一个Waardenburg综合征Ⅱ型家系SOX10基因的突变分析CSCD

目的对1例Waardenburg综合征Ⅱ型先证者及其家系成员进行SOX10基因的突变分析,探讨其可能的分子生物学致病原因。方法抽提先证者及其家系成员的外周血基因组DNA,芯片捕获高通量测序方法对MITF、PAX3、SOX10、SNA12、END3和ENDRB基因的全部外显子及其侧翼序列进行检测。根据高通量测序结果,对先证者及其父母进行突变位点的Sanger测序验证分析。结果Sanger测序结果显示先证者存在SOX10C．127c〉T（P．R43X）杂合突变,导致SOX10基因第43位编码精氨酸的密码子（CGA）突变为终止密码子（UGA）,产生截短蛋白,影响蛋白质功能的正常发挥。经检索人类基因突变数据库,该突变为未报道过的新突变。患儿父母未检测到该突变。结论先证者SOX10基因C．127c〉T（P．R43X）杂合突变可能是其分子生物学致病原因。     

Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing

Nephronophthisis (NPHP), an autosomal recessive kidney disease, is the most frequent genetic cause of chronic renal failure in the first three decades of life. Mutations in eight genes (NPHP1–8) have been identified. We here describe a combined approach for mutation screening of NPHP1, NPHP2, NPHP3, NPHP4, and NPHP5 in a worldwide cohort of 470 unrelated patients with NPHP. First, homozygous NPHP1 deletions were detected in 97 patients (21%) by multiplex PCR. Second, 25 patients with infantile NPHP were screened for mutations in inversin (NPHP2/INVS). We detected a novel compound heterozygous frameshift mutation (p.[Q485fs]+[R687fs]), and a homozygous nonsense mutation (p.R899X). Third, 37 patients presenting with NPHP and retinitis pigmentosa (Senior‐Løken syndrome [SLS]) were screened for NPHP5/IQCB1 mutations by direct sequencing. We discovered five different (three novel) homozygous premature termination codon (PTC) mutations (p.F142fsX; p.R461X; p.R489X; p.W444X; and c.488–1G>A). The remaining 366 patients were further investigated for mutations in NPHP1, NPHP3, and NPHP4. We applied a “homozygosity only” strategy and typed three highly polymorphic microsatellite markers at the respective loci. A total of 32, eight, and 14 patients showed homozygosity, and were screened by heteroduplex crude celery extract (CEL I) endonuclease digests. The sensitivity of CEL I was established as 92%, as it detected 73 out of 79 different known mutations simply on agarose gels. A total of 10 novel PTC mutations were found in NPHP1 (p.P186fs, p.R347X, p.V492fs, p.Y509X, and c.1884+1G>A), in NPHP3 (c.3812+2T>C and p.R1259X), and in NPHP4 (p.R59X, p.T1004fs, and p.V1091fs). The combined homozygosity mapping and CEL I endonuclease mutation analysis approach allowed us to identify rare mutations in a large cohort of patients at low cost. Hum Mutat 29(3), 418–426, 2008. © 2007 Wiley‐Liss, Inc.     

A novel nonsense mutation in TUSC3 is responsible for non-syndromic autosomal recessive mental retardation in a consanguineous Iranian family

The genetic basis of autosomal recessive mental retardation (ARMR) is extremely heterogeneous, and there is reason to suspect that the number of underlying gene defects may well go beyond 1,000. To date, however, only less than 10 genes have been implicated in non‐specific/non‐syndromic ARMR (NS‐ARMR). As part of an ongoing systematic study aiming to identify further ARMR genes, we investigated a consanguineous family with three patients with NS‐ARMR. By linkage analysis and subsequent mutation screening we identified a novel nonsense mutation (c.163C > T [p.Q55X]) in the second exon of the TUSC3 gene. This is the third MR causing defect in TUSC3 to be described and the second independent mutation in this gene in a cohort of more than 200 ARMR families from the Iranian population. This argues for a more prominent role of TUSC3 in the etiology of this genetically heterogeneous disorder as compared to most of the other so far identified ARMR genes. © 2011 Wiley‐Liss, Inc.     

Molecular Genetic Analysis of SLC3A1 and SLC7A9 Genes in Czech and Slovak Cystinuric Patients

Cystinuria is a frequently inherited metabolic disorder in the Czech population (frequency 1/5,600) caused by a defect in the renal transport of cystine and dibasic amino acids (arginine, lysine and ornithine). The disease is characterized by increased urinary excretion of the amino acids and often leads to recurrent nephrolithiasis. Cystinuria is classified into two subtypes (type I and type non‐I). Type I is caused predominantly by mutations in the SLC3A1 gene (2p16.3), encoding heavy subunit (rBAT) of the heterodimeric transporter. Cystinuria non‐I type is caused by mutations in the SLC7A9 gene (19q13.1). In this study, we present results of molecular genetic analysis of the SLC3A1 and the SLC7A9 genes in 24 unrelated cystinuria families. Individual exons of the SLC3A1 and SLC7A9 genes were analyzed by direct sequencing. We found ten different mutations in the SLC3A1 gene including six novel ones: three missense mutations (G140R), D179Y and R365P), one splice site mutation (1137‐2A>G), one deletion (1515_1516delAA), and one nonsense mutation (Q119X). The most frequent mutation, M467T; was detected in 36% of all type I classified alleles. In the SLC7A9 gene we found six mutations including three new ones: one missense mutation (G319R), one insertion (611_612insA) and one deletion (205_206delTG). One patient was compound heterozygote for one SLC3A1 and one SLC7A9 mutation. Our results confirm that cystinuria is a heterogeneous disorder at the molecular level, and contribute to the understanding of the distribution and frequency of mutations causing cystinuria in the Caucasian population.     

Generation and analysis of the thiazide‐sensitive Na+‐Cl− cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome

Gitelman syndrome (GS) is characterized by salt‐losing hypotension, hypomagnesemia, hypokalemic metabolic alkalosis, and hypocalciuria. To better model human GS caused by a specific mutation in the thiazide‐sensitive Na⁺‐Cl^? cotransporter (NCC) gene SLC12A3, we generated a nonsense Ncc Ser707X knockin mouse corresponding to human p.Ser710X (c.2135C>A), a recurrent mutation with severe phenotypes in Chinese GS patients. Compared with wild‐type or heterozygous littermates, homozygous (Hom) knockin mice fully recapitulated the phenotype of human GS. The markedly reduced Ncc mRNA and virtually absent Ncc protein expression in kidneys of Hom mice was primarily due to nonsense‐mediated mRNA decay (NMD) surveillance mechanisms. Expression of epithelial Na⁺ channel (Enac), Ca²⁺ channels (Trpv5 and Trpv6), and K⁺ channels (Romk1 and maxi‐K) were significantly increased. Late distal convoluted tubules (DCT) volume was increased and DCT cell ultrastructure appeared intact. High K⁺ intake could not correct hypokalemia but caused a further increase in maxi‐K but not Romk1 expression. Renal tissue from a patient with GS also showed the enhanced TRPV5 and ROMK1 expression in distal tubules. We suggest that the upregulation of TRPV5/6 and of ROMK1 and Maxi‐K may contribute to hypocalciuria and hypokalemia in Ncc Ser707X knockin mice and human GS, respectively. Hum Mutat 31:1–13, 2010. © 2010 Wiley‐Liss, Inc.     

A novel mutation of PANK4 causes autosomal dominant congenital posterior cataract

Though many mutations have been identified to be associated with the occurrence of congenital cataract, pathogenic loci in some affected families are still unknown. Clinical data and genomic DNA were collected from a four‐generation Chinese family. Candidate mutations were independently verified for cosegregation in the whole pedigree. Linkage analysis showed that the disease‐causing mutation was located between 1p36.21 and 1p36.33. Analysis of the whole‐exome sequencing data combined with linkage analysis identified a novel pathogenic variant (g.2451906C>T) at intron 4 of Pantothenate kinase 4 (PANK4 protein, PANK4 gene) in 1p36.32|606162. This variant showed complete cosegregation with the phenotype in the pedigree. The mutation was not detected in 106 normal controls nor in 40 sporadic congenital cataract patients. The mutation was demonstrated to significantly reduce the expression of the PANK4 protein level in the blood of cataract patients than that in normal individuals by ELISA. Pank4^?/? mice showed a cataract phenotype with increased numbers of apoptotic lens epithelial cells, fiber cell aggregation, and significant mRNA variation of crystallin family members. Thus, the association of a new entity of an autosomal dominant cataract with mutations in PANK4, which influences cell proliferation, apoptosis of lens epithelial cells, crystallin abnormalities, and fiber cell derangement, subsequently induces cataract.     

Spectrum of APC and MUTYH germ‐line mutations in Russian patients with colorectal malignancies

Distribution of cancer‐predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ‐line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early‐onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ‐line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next‐generation sequencing of 7 APC/MUTYH mutation‐negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low‐penetrant cancer‐associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH‐associated polyposis in Russia is likely to be 1:23 000.