Effect of type of screening laboratory on population-based occurrence of cervical lesions in Finland

Mutations in BRCA1 and BRCA2 genes confer a high risk of breast and ovarian cancer. As such, their identification is essential to reduce the risk of disease in healthy carriers, as well as in carriers who have already developed the disease because they are at increased risk for a second malignancy; moreover, noncarriers of BRCA1 and BRCA2 mutated families can be spared anxiety and unnecessary medical interventions. A number of problems, including large gene size, complex mutational spectra and genetic heterogeneity of the disease, however, make genetic testing labor intensive and often inconclusive. We devised a new mutation detection strategy called AGE (allele-specific gene expression) analysis that relies on the detection of a "functional effect" of the mutation at the RNA level known as "nonsense-mediated RNA decay," thus avoiding several of the problems of BRCA1 and BRCA2 genetic testing. In particular, (i) AGE analysis discriminates among the predisposing genes and identifies mutation carriers with a single RT-PCR reaction; (ii) it relies on the effect of truncating mutations, which represent the large majority of cases and thus identifies mutation carriers regardless of the specific genomic alteration; and (iii) it is specific for cis-regulatory mutations that are missed at present by most of the methods. As AGE analysis has the potential to identify most of the BRCA1 and BRCA2 mutation carriers, it can be used as a preliminary screening method, thereby accelerating and increasing the sensitivity of the genetic testing process. Notably, other hereditary diseases whose genetic analysis is hampered by similar problems could benefit from this kind of approach.     

Two novel mutations in the muscle glycogen phosphorylase gene in McArdle's disease

We report on a Spanish family with myophosphorylase (EC 2.4.1.1) deficiency (McArdle's disease). The proband and his symptomatic sister were compound heterozygous for two novel mutations: a T-to-G transversion in exon 14 (c1722 T>G) that changes a tyrosine to a stop codon (Y573X), and a G-to-A transition in exon 15 (c1827 G>A) that disrupts the consensus signal at the donor splicing site. These findings further expand knowledge of the genetic bases of muscle glycogen phosphorylase deficiency.     

A patient with peripheral demyelinating neuropathy,central dysmyelinating leukodystrophy,Waardenburg syndrome,and severe hypoganglionosis associated with a novel SOX10 mutation

In this report, we present the case of a female infant with peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease (PCWH) associated with a novel frameshift mutation (c.842dupT) in exon 5, the last exon of SOX10. She had severe hypoganglionosis in the small intestine and entire colon, and suffered from frequent enterocolitis. The persistence of ganglion cells made both the diagnosis and treatment difficult in the neonatal period. She also showed hypopigmentation of the irises, hair and skin, bilateral sensorineural deafness with hypoplastic inner year, severe demyelinating neuropathy with hypotonia, and diffuse brain hypomyelination. The p.Ser282GlnfsTer12 mutation presumably escapes from nonsense‐mediated decay and may generate a dominant‐negative effect. We suggest that hypoganglionosis can be a variant intestinal manifestation associated with PCWH and that hypoganglionosis and aganglionosis may share the same pathoetiological mechanism mediated by SOX10 mutations.     

Two frequent loss-of-function mutations in Aurora Kinase C gene in Algerian infertile men with macrozoospermia

Macrozoospermia is associated with severe male infertility. To date, the only gene implicated in this phenotype is the Aurora Kinase C gene. We report in this work the genetic screening of AURKC mutations in 34 patients with macrozoospermia among 3,536 Algerian infertile men. Nineteen patients (56%) were homozygotes for the c.144delC mutation, eight (23.52%) homozygotes for the c.744C>G (p.Y248*) mutation and two (5.88%) compound heterozygotes. No AURKC mutation was identified in five patients (14.7%). Interestingly and although it is generally accepted that nearly all positive mutated AURKC patients have close to 100% large-head spermatozoa, our results showed that 11 patients with AURKC mutations (32.35%) had large-headed spermatozoa lower than 70% (7 with c.144delC and 4 with p.Y248*), and no mutation was found in 2 patients who had >70% of macrocephalic spermatozoa. Twenty ICSI attempts were performed before genetic screening resulting in 39 embryos but no pregnancy was obtained. The sequencing of AURKC exons 3 and 6 is appropriate as a first-line genetic exploration in these patients to avoid unsuccessful ICSI attempts. A percentage of large head spermatozoa beyond 25% and a percentage of multiflagellar spermatozoa beyond 10% are predictive of a positive mutation diagnosis.     

Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII

BACKGROUND: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. OBJECTIVES: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. RESULTS: A FVII-green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII-GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X-FVII and p364X-FVII) were transfected and found to secrete low amounts of FVII (approximately 1% of Wt-FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X-FVII, 24 +/- 12 ng mL(-1), 3.6 +/- 0.8% of Wt-FVII activity; p364X-FVII, 26 +/- 10 ng mL(-1), 3.7+/-0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. CONCLUSIONS: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.     

一种新的FGA基因无义突变导致遗传性无纤维蛋白原血症

目的　对 1例遗传性无纤维蛋白原血症患者及其家系成员进行基因分析,探讨遗传性无纤维蛋白原血症发病的分子机制。方法　凝血酶法与免疫比浊法测定血浆纤维蛋白原 (Fg)含量,提取先证者及其家系成员外周血基因组DNA,PCR法扩增其Fg的FGA、FGB和FGG基因所有外显子和侧翼序列,DNA序列分析Fg的基因异常。将先证者突变序列、家系成员和 50名正常人相应序列的PCR产物用限制性内切酶RsaⅠ消化,以进一步确定基因突变位点并排除基因多态性。结果　用Clauss法检测不到先证者及其父亲的血浆Fg,用免疫比浊法测定时,Fg含量均<0. 02g/L。两人FGA基因外显子 4第 3108位核苷酸发生C→T纯合性改变,使Fg的Aα链第 150位密码子 (CAG,编码Gln)突变为终止密码TAG。先证者及其父亲的FGA基因外显子 4和侧翼序列的PCR产物,不能被RsaⅠ酶切,其母亲及部分家系成员的PCR产物被部分酶切,而正常人和该家系中的 5个成员的PCR产物可被完全酶切。结论　FGA基因(外显子 4)Q150X无义突变导致该家系先证者及其父亲遗传性无Fg血症,家系中部分成员为携带者,此突变是一种国际上尚未报道的新的突变类型。     

Severe juvenile retinoschisis associated with a 33-bps deletion in XLRS1 gene

X-linked juvenile retinoschisis is a form of vitreoretinal dystrophy that is characterized by foveal and peripheral splitting of the retinal nerve fiber layer. Pathognomonic of this disorder is a microcystic radiate appearance in the fovea. We encountered a 10-year-old, mildly retarded, Japanese boy, who exhibited a widely extended macular retinoschisis bilaterally. A break in the inner layer of the left eye mimicked a lamellar macular hole, which is a rare manifestation of the disease. Peripheral retinoschisis was absent. Only a few reports have described marked bilateral macular retinoschisis that involved the entire posterior pole, while various other macular findings have been reported. This patient with a severe form of retinoschisis was found to harbor the deletion of 33 base pairs, including the boundary region of exon 3 and intron 3 in the XLRS1 gene.     

凝血因子ⅩⅢA基因Arg77Cys错义突变和Arg174stop无义突变致凝血因子ⅩⅢ缺陷的分子机制

本研究以凝血因子ⅩⅢ(FⅩⅢ)基因突变为研究的切入点,从分子水平研究这些突变致病的机制.构建野生型FⅩⅢA重组表达质粒,用定点突变方法获得含有2种突变(Arg77Cys及Arg174stop)的FⅩⅢA重组表达质粒.分别将上述重组质粒通过脂质体介导的基因转移方法转染到COS-7细胞表达;用发色底物法检测转染细胞中FⅩⅢ...