Three new adenosine deaminase mutations that define a splicing enhancer and cause severe and partial phenotypes: implications for evolution of a CpG hotspot and expression of a transduced ADA cDNA

We report three novel adenosine deaminase (ADA) mutations withinteresting implications. A Somali child with severe combinedimmunodeficiency disease (SCID) had reduced ADA mRNA in T cellsand was homozygous for the nonsense mutation Q3X. Unexpectedly,her healthy father was a compound ADA heterozygote whose secondallele carried a ‘partial’ mutation, R142Q, dueto a GA transition of a CpG dinucleotide. A CT transition ofthe same CpG produced a nonsense mutation, R142X, in two homozygousCanadian Mennonite infants with SCID. The severe and healthyphenotypes associated with R142X and R142Q, the high frequencyof ‘partial’ ADA mutations arising from CpGs inhealthy individuals of African descent and the presence of CAA(glutamine) at codon 142 in murine ADA, suggest selection forreplacement of this CpG hotspot by CpA during ADA evolution.R142X, located within a purine-rich segment at nt 62/116 ofexon 5, caused skipping of the exon, possibly by disruptinga splicing enhancer. Absence of exon 5 in T cell ADA mRNA andlow ADA activity in T cells and erythrocytes obtained at age18–22 months from one of the Mennonite children, indicatelimited expression of a normal ADA cDNA from retrovirally transducedCD34+ umbilical cordleukocytes infused shortly after birth inan attempt at stem cell gene therapy.     

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g相似文献   

Molecular study of WISP3 in nine families originating from the Middle-East and presenting with progressive pseudorheumatoid dysplasia: identification of two novel mutations, and description of a founder effect

Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive syndrome characterized by the presence of spondyloepiphyseal dysplasia associated with pain, stiffness, and swelling of multiple joints, osteoporosis, and the absence of destructive bone changes. The disorder is caused by mutations of the WISP3 gene located on chromosome 6q22. We hereby report the molecular study of the WISP3 gene in nine unrelated consanguineous families originating from the Middle-East: three from Lebanon, five from Syria, and one from Palestinian Bedouin descent, all affected with PPD. Five different sequence variations were identified in the WISP3 gene, two of them being new mutations: the c.589G --> C transversion at codon 197, responsible for a splicing defect (A197fsX201); and the c.536_537delGT deletion (C179fsX), both in exon 3. In all other families, the affected patients were homozygous for a previously described nonsense mutation, namely c.156C --> A (C52X). Interestingly, in the latter families, the C52X mutation was always found associated with a novel c.248G --> A (G83E) variation, suggesting the existence of a founder effect.     

Molecular characterization of the putative T-cell receptor cavity of the superantigen staphylococcal enterotoxin B.

A number of investigators have utilized a variety of methods to identify the structural basis for the interaction of superantigens with the T-cell receptor beta-chain. The previous studies strongly suggest that a region of the toxin near residues N23, Y61, Y91 and D209 is important for this binding activity. Examination of crystal structure data shows that these residues line the rim of one side of a shallow cavity in the toxin. In an attempt further to define the face of the staphylococcal enterotoxin B (SEB) molecule involved in the interaction with the beta-chain, we have employed a polymerase chain reaction (PCR)-based, site-specific mutagenesis method to generate amino acid substitutions of residues on the opposite side of this putative T-cell receptor interaction cavity. Our results show that Y175 and N179 appear to be involved in the function of this superantigen, since each of several substitutions at this position exhibits a significantly reduced ability to induce T-cell proliferation. At the same time, mutation of the proximal Y186 does not alter the superantigen activity of SEB. Binding analysis of these mutants shows that class II binding activity is not significantly altered. Analysis of the responding T cells shows that the mutant toxins maintain T-cell receptor V beta selectivity. However, responses of T cells bearing the V beta 8.1 allele appear to be particularly diminished. When viewed in the context of other results reported in the literature, our results suggest that the T-cell receptor interaction site involves SEB residues which ring both the Y175/N179-side and the N23-side of a cavity on one side of the toxin molecule.     

Impact of influenza A virus neuraminidase mutations on the stability, activity, and sensibility of the neuraminidase to neuraminidase inhibitors.

BACKGROUND: The influenza neuraminidase plays a critical role in the spread of the influenza A and B viruses. Through the cleavage of terminal sialic acid from glycoconjugates, it facilitates the elution of progeny virions from infected cells and prevents their self-aggregation. OBJECTIVES: Our objective was to study the impact of mutations at framework sites not under direct selective pressure in the neuraminidase active site. STUDY DESIGN: In the A/Moscow/10/99 (H3N2) virus background, viruses containing mutations in NA framework residues (E119D, R156K, W178L, S179A, D198N, I222L, E227G, H274Y, E277G, N294D, and E425G) were constructed by reverse genetics. After several passages on MDCK cells, fluorimetric assays were conducted to assess the neuraminidase activity and sensibility to the neuraminidase inhibitors (IC50). RESULTS: Among the viruses detectable through the phenotypic tests, R156K, I222L, H274Y, N294D and E425G viruses presented a NA activity between 70% and 100% of the A/Moscow/10/99 wild type one. The D198N and the E119D mutations decreased seriously in NA activity compared to the wild-type (>10-fold). The I222L mutation reduced susceptibility to oseltamivir (18-fold). CONCLUSION: With the exception of one mutation, framework mutations on N2 background do not induce resistance. Nevertheless they tend to decrease slowly the sensitivity to one or the other inhibitors.     

PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症

目的对一先天性无虹膜症家系进行了致病基因PAX6的突变分析。方法PCR反应扩增PAX6基因的所有外显子,PCR产物进行SSCP（单链构象多态性）分析,通过患者与正常人带型的差异来确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接测序找到突变位点。PCR产物进一步亚克隆到pGEM-T载体,测序验证突变位点。结果发现基因突变为PAX6基因第2内含子和第3外显子之间的剪接识别位点＂AG＂中的碱基A的丢失（IVS2-2delA）。结沦PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症。     

R179H mutation in ACTA2 expanding the phenotype to include prune-belly sequence and skin manifestations

Mutations in ACTA2 (smooth muscle cell-specific isoform of α-actin) lead to a predisposition to thoracic aortic aneurysms and other vascular diseases. More recently, the ACTA2 R179H mutation has been described in individuals with global smooth muscle dysfunction. We report a patient heterozygous for the mutation in ACTA2 R179H who presented with megacystis at 13 weeks gestational age and, at birth, with prune-belly sequence. He also had deep skin dimples and creases on his palms and soles, a finding not previously described but possibly related to ACTA2. To our knowledge, this is the first report of the R179H mutation in ACTA2 in a child with prune-belly sequence. We think the R179H mutation in ACTA2 should be included in the differential diagnosis of individuals presenting with the sequence without an identified mechanical obstruction. Furthermore, as ACTA2 R179H has been reported in patients with severe vasculomyopathy and premature death, we recommend that molecular testing for this mutation be considered in fetuses presenting with fetal megacystis with a normal karyotype, particularly if the bladder diameter is 15?mm or more, to allow expectant parents to make an informed decision.     

Metachromatic leucodystrophy: a newly identified mutation in arylsulphatase A,D281Y,found as a compound heterozygote with I179L in an adult onset case

The majority of mutations identified in patients with Metachromatic leucodystrophy are unique to individual families. We report here a new mutation in the arylsulphatase A gene (D281Y) identified in a patient with late‐onset Metachromatic leucodystrophy. This mutation was inherited in cis with the common pseudo‐deficiency allele and in trans with the previously described I179S (250100.0008) mutation which complicated the enzymatic diagnosis of this condition. Sequence comparison shows D281 to be highly conserved amongst the arylsulphatases. The clinical features of this patient which are predominantly of a slowly progressive psychiatric and intellectual deterioration rather than rapid neurological impairment are typical of I179S compound heterozygotes. Hum Mutat 14:447, 1999. ©1999 Wiley‐Liss, Inc.     

Luc7p, a novel yeast U1 snRNP protein with a role in 5′ splice site recognition

The characterization of a novel yeast-splicing factor, Luc7p, is presented. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors. We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth. The composition of yeast U1 snRNP is altered in luc7 mutant strains. Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains. These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition.     

Novel mutations in African American patients with glycogen storage disease Type II. Mutations in brief no. 209. Online

The infantile form of GSD II (an inherited deficiency of the lysosomal enzyme, acid alpha-glucosidase, Pompe disease) is a severe and invariably fatal disease characterized by a rapidly progressive generalized hypotonia, hepatomegaly, and cardiomegaly. We have recently demonstrated that African American patients share a common nonsense R854X mutation in exon 18 (Becker et al., 1998). Two other mutations, D645E and M519V, have been identified in individual African American patients (Hermans et al., 1993a; Huie et al., 1994a). We describe here three novel mutations in this population group: a missense W481R in exon 10, a deletion of a T1441 in exon 10, and a splicing defect at the 5' donor site of intron 8 (IVS g+la) . The splicing defect is shared by two unrelated patients and it is linked to intragenic polymorphic sites identical to those found in patients bearing the common R854X mutation.     

新疆汉族、维吾尔族HPV16E6基因变异与子宫颈癌及宫颈病变的关系

目的探讨新疆地区汉族、维吾尔族宫颈癌及宫颈病变中HPV16型E6基因变异分布及频率,比较两个民族之间的差异。方法对140例HPV16阳性标本,设计E6基因特异性引物,通过PCR扩增HPV16E6全长基因,PCR产物直接测序,进行序列分析并与德国标准株对比,筛选突变位点。结果 123例汉族、维吾尔族妇女宫颈癌及宫颈病变标本中均存在HPV16E6变异株,其中突变率分别为:47.37%(27/57)和50%(33/66),E6突变位点主要是L83V、D25E、D64E、I73V、H78Y、D113E,其中L83V变异株在两个民族突变中占的比例均最高,汉族L83V突变频率为29.82%(17/57)显著低于维吾尔族的40.90%(27/66)(P0.05)。而汉族D25E突变频率为19.30%(11/57)显著高于维吾尔族的7.58%(5/66)(P0.05),D64E变异株在维族突变株中占的比例为6.1%,而汉族标本中未检出。结论两个民族HPV16 E6基因变异发生的位置和变异发生的频率存在差异,维吾尔族人群中HPV16E6基因D64E变异株可能与该地区维吾尔族宫颈癌高发存在一定的关系。     

在中国人先天性厚甲症Ⅰ型患者中检测出两种KRT6A基因突变

目的 分别研究一个先天性厚甲症Ⅰ型家系和一个散发患者基因突变,探讨基因型与表型的相关性.方法 研究对象包括6例先天性厚甲症Ⅰ型患者,其中包括家系中5例患者和1例散发病例,同时取家系中1名正常人及与该家系无关的100名正常人作为正常对照排除多态性,采用长链PCR特异扩增了外周血基因组DNA的KRT16和KRT6A两个候选致病基因全长,PCR产物直接测序检测突变.结果 家系和散发病例的KRT16基因均未发现基因突变.而两者的KRT6A基因分别出现了突变:家系中5例患者的第465位密码子由TAC突变为CAC,导致酪氨酸由组氨酸替代(Y465H);散发患者KRT6A基因第171位密码子由AAC突变为GAC,导致天门冬酰胺由天门冬氨酸替代(N171D),而该家系中的1名正常人及与该家系无关的100名正常人的DNA测序结果未发现此突变.Y465H和N171D分别位于2B的终末端和1A的起始部这两个突变热点.结论 该家系和散发病例分别存在KRT6A基因突变Y465H和N171D,其中前者为一新的错义突变,N171D近期已有文献报道.该突变可能为先天性厚甲症Ⅰ型的遗传病因.     

Neutralisation and binding of VHS virus by monovalent antibody fragments.

We have previously reported the cloning and characterisation of the heavy and light chain variable domain genes encoding three monoclonal antibodies (Mabs) that bind viral haemorrhagic septicaemia virus (VHSV). Two of these antibodies, 3F1H10 and 3F1A2 both neutralised the virus though 3F1A2 appeared to recognise a broader range of virus isolates. The variable domains of these two antibodies differ by only four residues (Lorenzen et al., 2000a. Fish Shellfish Immunol. 10, 129-142). To further study the mechanism of neutralisation, Fab fragments as well as a series of recombinant bacterial single chain antibody (scAb) fragments were generated from the three anti-VHSV Mabs and their variable domain genes, respectively. Fabs and scAbs derived from the neutralising Mabs were both able to neutralise the VHSV type 1 isolate DK-F1. In addition, a series of scAb fragments were produced using the 3F1H10 variable heavy (VH) chain and variable light (Vkappa) chain domains but containing, either alone or in dual combination, each of the four different residues present in 3F1A2. The dissociation constants of Mabs 3F1H10 and 3F1A2 and their respective Fab and scAb fragments were measured by BIAcore analysis and found to correlate with the capacity of each molecule to neutralise DK-F1. These investigations, together with computer assisted molecular analysis of the theoretical influence of each mutation on antigen binding, led to the identification of a single mutation at position 35a in the VH domain as having the most marked impact on viral neutralisation.     

Novel donor splice site mutation of ABCG5 gene in sitosterolemia

In a patient with sitosterolemia, we found two different mutations of the ATP-binding cassette, subfamily G, member 5 (ABCG5) gene. The first is a missense mutation that changes the amino acid residue at position 419 from arginine to histidine, i.e., R419H. The second is a novel splicing mutation affecting the invariant guanine at the first base of the donor splice site of intron 12, i.e., IVS12 + 1G --> A. The father of the patient is heterozygous for the missense mutation, and the mother is heterozygous for the splicing mutation. No mutations were found in the sister of the patient. Up until now, the missense mutation has only been found in Japanese patients with sitosterolemia. We believe that R419H in our Chinese patient may have the same origin as the mutation in the Japanese patients with sitosterolemia.     

Bruton's tyrosine kinase mutations in 8 Chinese families with X-linked agammaglobulinemia

Bruton's tyrosine kinase (BTK) is involved in B-cell development. Mutation of BTK results in X-linked agammaglobulinemia (XLA). BTK is expressed in most haemopoietic lineages except mature T cells and plasma cells. We identified six novel and two known mutations of BTK in 11 Chinese XLA patients from 8 families. Family 1 had a novel point mutation at the start codon (135G-->T) in exon 2. Family 2 had known mutation of single A insertion in a stretch of 7 A residues (341-347insA) recognized as mutation hotspot in exon 3. Family 3 had a novel point mutation in exon 11 (1074A-->G) which led to aberrant splicing. Family 4 had known mutation in exon 19 (2053C-->T) in CpG mutation hotspot. The novel mutation of family 5 was an A deleted in a run of three As (1017-1019delA) in exon 10. In family 6, exons 2 and 3 were lost in BTK mRNA, a novel deletion. Family 7 had a novel substitution in exon 2 (227T-->C) which led to change of a conserved leucine to serine. Family 8 had a novel point mutation at beginning of intron 14 (IVS14+ 6 T-->G) resulting in aberrant splicing. Hum Mutat 15:385, 2000.