Search

In clinical practice, the distinction between type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) can be challenging, leaving patients with “ambiguous” diabetes type. Insulin-treated patients (n=115) previously diagnosed with T2DM had to be re-classified based on clinical phenotype and laboratory results, and were operationally defined as having an ambiguous diabetes type. They were compared against patients with definite T1DM and T2DM regarding 12 clinical and laboratory features typically different between diabetes types. Characteristics of patients with ambiguous diabetes type, representing approximately 6% of all patients with T1DM or T2DM seen at our specialized clinic, fell in between those of patients with definite T1DM and T2DM, both regarding individual features and with respect to a novel classification based on multi-variable regression analysis (P

ASTM G67 is the standard test method for determining the susceptibility to intergranular corrosion (IGC) of 5xxx series aluminum alloys and requires measuring mass loss after exposure to nitric acid (the nitric acid mass loss test, NAMLT). This mass loss, and thus the IGC susceptibility, is dependent on orientation of the wrought product, but the quantitative degree of this dependence has not been well studied or documented. The orientation dependence of IGC susceptibility for AA5083 (UNS A95083) in H131 and H116 tempers is quantitatively presented herein. The underlying mechanism behind this dependence is also explored. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article about the use of the nucleotide excision repair (NER) to fix ultraviolet (UV)-induced photoproducts associated with gene mutations by O. D. Schärer in the previous issue.

Only 16 XPG-defective patients with 20 different mutations have been described. The current hypothesis is that missense mutations impair repair (xeroderma pigmentosum (XP) symptoms), whereas truncating mutations impair both repair and transcription (XP and Cockayne syndrome (CS) symptoms). We identified three cell lines of XPG-defective patients (XP40GO, XP72MA, and XP165MA). Patients' fibroblasts showed a reduced post-UVC cell survival. The reduced repair capability, assessed by host cell reactivation, could be complemented by XPG cDNA. XPG mRNA expression of XP165MA, XP72MA, and XP40GO was 83%, 97%, and 82.5%, respectively, compared with normal fibroblasts. XP165MA was homozygous for a p.G805R mutation; XP72MA and XP40GO were both compound heterozygous (p.W814S and p.E727X, and p.L778P and p.Q150X, respectively). Allele-specific complementation analysis of these five mutations revealed that p.L778P and p.W814S retained considerable residual repair activity. In line with the severe XP/CS phenotypes of XP72MA and XP165MA, even the missense mutations failed to interact with the transcription factor IIH subunits XPD and to some extent cdk7 in coimmunoprecipitation assays. Immunofluorescence techniques revealed that the mutations destabilized early recruitment of XP proteins to localized photodamage and delayed their redistribution in vivo. Thus, we identified three XPG missense mutations in the I-region of XPG that impaired repair and transcription and resulted in severe XP/CS.

Patients belonging to xeroderma pigmentosum (XP) complementation group C comprise one-third of all XP patients. Only four major reports compiled larger groups of XP-C patients from southern Europe (12 pts), North America (16 pts) and Africa (14 and 56 pts) as well as their genetic background (46 XPC mutations). We identified 16 XP-C patients from Germany. Interestingly, only five patients exhibited severe sun sensitivity. The mean age of XP diagnosis was 9.4 years, and the median age of the first skin cancer was 7 years. Neurological symptoms were absent in all but two patients. Primary fibroblasts from all 16 patients showed reduced post-UV cell survival (mean: 50% vs 93% in normal cells) and reduced reactivation of an UV-treated luciferase reporter gene (mean: 6.4% vs 30.7% in normal cells). XPC mRNA expression was also greatly reduced compared with normal cells (mean: 14.3%; range 8.3-25.7%) except in XP47MA (274.1%). All patients carried homozygous XPC mutations. Four mutations have been described previously: c.1747_1748delTG (found in 4/16), c.567 C>T (4/16), c.1839 C>T (1/16) and a complex insertion/deletion mutation in exon 9 (1/16). The novel frameshift mutations c.446_447delAG (2/16), c.1525insA (1/16) and c.2271delC (1/16) lead to truncated XPC proteins as does the novel nonsense mutation c.843C>T (1/16). XP47MA carries an interesting mutation (c.2538_2540delATC; p.Ile812del) resulting in an in-frame single amino acid deletion. This mutation results in a classical XP phenotype, a non-functional XPC protein, but elevated XPC mRNA expression. Our study indicates that extrinsic factors may contribute to XP-C symptom severity due to nonsense-mediated message decay., (© 2012 John Wiley & Sons A/S.)

Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner., (© 2011 John Wiley & Sons A/S.)

Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV-induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV-induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post-UV cell survival of cyclosporin A- and everolimus-treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine-6,4-pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post-UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus-treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A-induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A-treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling-dependent impairment of NER., (© 2010 John Wiley & Sons A/S.)

We examined the clinical, molecular and genetic features of a 16-year-old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2 years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger-tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post-UV cell survival (D(37) = 3.8 J/m(2)), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.

Xeroderma pigmentosum (XP) patients exhibit a 1000-fold increased risk for developing skin cancers including malignant melanoma. We investigated the role of three variant alleles of the DNA repair gene XPC and one variant allele of the XPG gene in a hospital-based case-control study of 294 Caucasian patients from Germany with malignant melanoma and 375 healthy control individuals from the same area matched by sex. The polymorphisms G1580A (XPC exon 8; Arg492His), T1601C (XPC exon 8; Val499Ala), G2166A (XPC exon 10; Arg687Arg), and C3507G (XPG exon 15; Asp1104His) were not in linkage disequilibrium. The allele frequencies (cases: controls) were for 1580A 6.29%: 5.63%, for 1601C 79.08%: 78.28%, for 2166A 26.19%: 28.13%, and for 3507G 79.86%: 78.61%. We found no association of the homozygous 1580A, 1601C, 2166A, and 3507G genotypes with increased risks of melanoma: OR 1.254 (95% CI: 0.486-3.217), OR 1.108 (95% CI: 0.629-1.960), OR 0.817 (95% CI: 0.490-1.358), and OR 1.168 (95% CI: 0.670-2.044), respectively. Exploratory analyses of subgroups of melanoma patients compared to all controls indicated no association of these genotypes with increased risks for development of multiple primary melanomas (n = 28), a negative family history for melanoma (n = 277), melanomas in individuals with a low number of nevi (n = 273), melanomas in individuals older than 55 years (n = 142), and melanomas thicker than 1 mm (n = 126).

Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Pyruvate kinase (PK) deficiency (PKD) is an autosomal recessive disorder with the typical manifestation of nonspherocytic hemolytic anemia. We analyzed the mutant enzymes of 10 unrelated patients with PKD, whose symptoms ranged from a mild, chronic hemolytic anemia to a severe anemia, by sequence analysis for the presence of alterations in the PKLR gene. In all cases the patients were shown to be compound heterozygous. Eight novel mutations were identified: 458T-->C (Ile153Thr), 656T-->C (Ile219Thr), 877G-->A (Asp293Asn), 991G-->A (Asp331Asn), 1055C-->A (Ala352Asp), 1483G-->A (Ala495Thr), 1649A-->T (Asp550Val), and 183-184ins16bp. This 16 bp duplication produces a frameshift and subsequent stop codon resulting in a drastically reduced mRNA level, and probably in an unstable gene product. Surprisingly, the existence of M2-type PK could be demonstrated in the patient's red blood cells. The study of different polymorphic sites revealed, with one exception, a strict linkage of the 1705C, 1738T, IVS5(+51)T, T(10) polymorphisms and the presence of 14 ATT repeats in intron 11. Our analyses show the consequences of a distorted structure on enzyme function and we discuss the correlations between the mutations identified and the parameters indicative for enzyme function., (Copyright 2000 Wiley-Liss, Inc.)

Using direct sequencing we analysed the pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia due to PK deficiency. A novel promoter mutation -249delA relative to the translation initiation site and the common 1529A mutation in exon 11 of the gene could be identified. Reverse transcription (RT)-PCR analysis combined with restriction digestion revealed that the -249delA mutation leads to a reduction in the amount of mRNA produced from this allele to about 6% of normal. We assume that both mutations would account for the PK deficiency in the compound heterozygous patient.