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Choroideremia is an X-linked inherited retinal disorder (IRD) characterized by the degeneration of retinal pigment epithelium, photoreceptors, choriocapillaris and choroid affecting males with variable phenotypes in female carriers. Unlike other IRD, characterized by a large clinical and genetic heterogeneity, choroideremia shows a specific phenotype with causative mutations in only one gene, CHM. Ongoing gene replacement trials raise further interests in this disorder. We describe here the clinical and genetic data from a French cohort of 45 families, 25 of which carry novel variants, in the context of 822 previously reported choroideremia families. Most of the variants represent loss-of-function mutations with eleven families having large (i.e. ≥6 kb) genomic deletions, 18 small insertions, deletions or insertion deletions, six showing nonsense variants, eight splice site variants and two missense variants likely to affect splicing. Similarly, 822 previously published families carry mostly loss-of-function variants. Recurrent variants are observed worldwide, some of which linked to a common ancestor, others arisen independently in specific CHM regions prone to mutations. Since all exons of CHM may harbor variants, Sanger sequencing combined with quantitative polymerase chain reaction or multiplex ligation-dependent probe amplification experiments are efficient to achieve the molecular diagnosis in patients with typical choroideremia features.

Membrane transporters influence biological functions in the ocular lens. Here, we investigate the monocarboxylate transporter 12 (MCT12), also called creatine transporter 2 (CRT2), which is found in the ocular lens and is involved in cataract. As the age-related form affects about half of the population world-wide, understanding relevant pathomechanisms is a prerequisite for exploring non-invasive treatments. We screened the coding exons of the gene SLC16A12 in 877 patients from five cohorts, including Caucasian and Asian ethnicities. A previously identified risk factor, SNP rs3740030, displayed different frequencies in the Asian cohorts but risk could not be established. In 15 patients 13 very rare heterozygous nucleotide substitutions were identified, of which eight led to non-synonymous and four to synonymous amino acid exchanges and one mapped to the canonical splice site in intron 3. Their impact on creatine transport was tested in Xenopus laevis oocytes and human HEK293T cells. Four variants (p.Ser158Pro, p.Gly205Val, p.Pro395Gln and p.Ser453Arg) displayed severe reduction in both model systems, indicating conserved function. Two of these, p.Gly205Val, and p.Ser453Arg, did not localize to the oocyte membrane, suggesting possible impacts on protein interactions for transporter processing. In support, exogenously supplied excess of MCT12's chaperone CD147 in HEK293T cells led to a partial recovery of the defective uptake activity from p.Gly205Val and also from mutant p.Pro395Gln, which did localize to the membrane. Our findings provide first insight in the molecular requirements of creatine transporter, with particular emphasis on rescuing effects by its chaperone CD147, which can provide useful pharmacological information for substrate delivery.

Item does not contain fulltext Immunodeficiency with centromeric instability and facial anomalies (ICF) syndrome is a primary immunodeficiency, predominantly characterized by agammaglobulinemia or hypoimmunoglobulinemia, centromere instability and facial anomalies. Mutations in two genes have been discovered to cause ICF syndrome: DNMT3B and ZBTB24. To characterize the clinical features of this syndrome, as well as genotype-phenotype correlations, we compared clinical and genetic data of 44 ICF patients. Of them, 23 had mutations in DNMT3B (ICF1), 13 patients had mutations in ZBTB24 (ICF2), whereas for 8 patients, the gene defect has not yet been identified (ICFX). While at first sight these patients share the same immunological, morphological and epigenetic hallmarks of the disease, systematic evaluation of all reported informative cases shows that: (1) the humoral immunodeficiency is generally more pronounced in ICF1 patients, (2) B- and T-cell compartments are both involved in ICF1 and ICF2, (3) ICF2 patients have a significantly higher incidence of intellectual disability and (4) congenital malformations can be observed in some ICF1 and ICF2 cases. It is expected that these observations on prevalence and clinical presentation will facilitate mutation-screening strategies and help in diagnostic counseling. 01 november 2013

Nature Communications 6: Article number: 7870 (2015); Published: 28 July 2015; Updated: 22 June 2016 In Fig. 1b of this Article, the sex of patient 2.1 in family A is incorrect, and should be depicted as male. The correct version of this figure appears below. Figure 1

Patients with the very rare eye pathology Wagner disease (OMIM #143200) present with an abnormal (empty) vitreous, retinal detachment and altered electroretinogram (ERG). The disease is progressive and can eventually lead to blindness. No therapy can be offered to date. The genetic basis is the presence of mutations in the VCAN gene, encoding the large extracellular matrix molecule versican, which is a component of the vitreous. All identified mutations map to the canonical splice sites flanking exon 8, resulting in low number of aberrant splice products and a severe increase in two (V2, V3) of the four naturally occurring splice variants. The pathomechanism of Wagner's disease is poorly understood and a mouse model may afford further insight. The hdf -/- mice, named for their initial phenotype of heart defects, carry a null allele for Vcan that leads to embryonic lethality when homozygous, but heterozygote animals are viable. Here we investigated a possible eye phenotype in the heterozygous animals. While the overall morphology of retina and ciliary body appears to be normal, older (17 months) mutant animals show a decrease in ERG signaling profiles affecting the a-, b- and c-waves. This aspect of altered ERG profile demonstrates similarities to the human disease manifestation and underlines the suitability of heterozygous hdf+/- mice as a model for Wagner disease.

During the last two to three decades, a large body of work has revealed the molecular basis of many human disorders, including retinal and vitreoretinal degenerations and dysfunctions. Although belonging to the group of orphan diseases, they affect probably more than two million people worldwide. Most excitingly, treatment of a particular form of congenital retinal degeneration is now possible. A major advantage for treatment is the unique structure and accessibility of the eye and its different components, including the vitreous and retina. Knowledge of the many different eye diseases affecting retinal structure and function (night and colour blindness, retinitis pigmentosa, cone and cone rod dystrophies, photoreceptor dysfunctions, as well as vitreoretinal traits) is critical for future therapeutic development. We have attempted to present a comprehensive picture of these disorders, including biological, clinical, genetic and molecular information. The structural organization of the review leads the reader through non-syndromic and syndromic forms of (i) rod dominated diseases, (ii) cone dominated diseases, (iii) generalized retinal degenerations and (iv) vitreoretinal disorders, caused by mutations in more than 165 genes. Clinical variability and genetic heterogeneity have an important impact on genetic testing and counselling of affected families. As phenotypes do not always correlate with the respective genotypes, it is of utmost importance that clinicians, geneticists, counsellors, diagnostic laboratories and basic researchers understand the relationships between phenotypic manifestations and specific genes, as well as mutations and pathophysiologic mechanisms. We discuss future perspectives.

X-linked Norrie disease, familial exudative vitreoretinopathy (FEVR), Coat's disease and retinopathy of prematurity are severe human eye diseases and can all be caused by mutations in the Norrie disease pseudoglioma gene. They all show vascular defects and characteristic features of retinal hypoxia. Only Norrie disease displays additional neurological symptoms, which are sensorineural hearing loss and mental retardation. In the present study, we analysed transcript levels of the ligand Norrin (Ndph) and its two receptors Frizzled-4 (Fzd4) and LDL-related protein receptor 5 (Lrp5) in six different brain regions (cerebellum, cortex, hippocampus, olfactory bulb, pituitary and brain stem) of 6- to 8-month-old wild-type and Ndph knockout mice by quantitative real-time PCR. No effect of the Ndph knockout allele on Fzd4 or Lrp5 receptor expression was found. Furthermore, no alterations of the transcript levels of three hypoxia-regulated angiogenic factors (Vegfa, Itgrb3 and Tie1) were observed in the absence of Norrin. Interestingly, we identified significant differences in Ndph, Fzd4 and Lrp5 transcript levels in brain regions of wild-type mice and observed highest expression of Norrin and frizzled-4 in cerebellum. Transcript analyses were correlated with morphological data obtained from cerebellum and immunohistochemical studies of blood vessels in different brain regions. Vessel density was reduced in the cerebellum of Ndph knockout mice but the number of Purkinje and granular cells was not altered. This provides the first description of a brain phenotype in Ndph knockout mice, which will help to elucidate the role of Norrin in the brain.

Unobstructed vision requires a particular refractive index of the lens, a measure based on the organization of the structural proteins within the differentiated lens cells. To ensure an intact lens structure, homeostasis within the lens cells is indispensable. Alterations of the lens structure result in opacity and lead to cataract. Renal glucosuria is defined by elevated glucose level in the urine without hyperglycemia and without evidence of morphological renal anomalies. In a Swiss family with autosomal dominant juvenile cataract, microcornea, and renal glucosuria, we have identified a nonsense mutation in a member of the carboxylic acid transporter family SLC16. The underlying gene defect in SLC16A12 resides within a 3 cM region on chromosome 10q23.13 defined by linkage mapping of this phenotype. We found tissue-specific variability of SLC16A12 transcript levels in control samples, with high expression in the eye and kidney, the two organs affected by this syndrome. This report demonstrates biological relevance of this solute carrier. We hypothesize that SLC16A12 is important for lens and kidney homeostasis and discuss its potential role in age-related cataract.

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo. However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

The life-threatening Immunodeficiency, Centromeric Instability and Facial Anomalies (ICF) syndrome is a genetically heterogeneous autosomal recessive disorder. Twenty percent of patients cannot be explained by mutations in the known ICF genes DNA methyltransferase 3B or zinc-finger and BTB domain containing 24. Here we report mutations in the cell division cycle associated 7 and the helicase, lymphoid-specific genes in 10 unexplained ICF cases. Our data highlight the genetic heterogeneity of ICF syndrome; however, they provide evidence that all genes act in common or converging pathways leading to the ICF phenotype., Immunodeficiency-centromeric instability-facial anomalies syndrome is a life threatening autosomal recessive disorder caused by mutations in DNMT3B and ZBTB24. Here Thijssen et al. identify mutations in CDCA7 and HELLS in previously unexplained cases.

Retinal dystrophies constitute a group of clinically and genetically heterogeneous diseases that cause visual impairment. As treatments are not readily available, readout assays performed in patient-derived cells can aid in the development and comparative analysis of therapeutic approaches. We describe a new method with which the localization of the retinitis pigmentosa GTPase regulator (RPGR) protein along the cilium can be used as a measure for treatment efficacy. In a patient-derived fibroblast cell line, we found that the RPGR protein is mislocalized along the ciliary axoneme. The patient carried a point mutation that leads to skipping of RPGR exon 10. We confirmed that this skipping is causative for the impaired localization of RPGR using a U7 small nuclear RNA (U7snRNA)-based antisense approach in control cells. Treatment of the patient-derived fibroblasts with therapeutic U1snRNA significantly corrected the proteins' mislocalization. In this proof of principle study, we show that detecting the RPGR protein along the cilium provides a reliable and quantifiable readout assay to evaluate the efficacy of therapies intended to correct or silence RPGR gene mutations. This method opens the possibility to compare different therapeutic agents, and thus facilitate the identification of treatment options for the clinically and molecularly complex RPGR-associated diseases.

Mutations in genes encoding either components of the phototransduction cascade or proteins presumably involved in signaling from photoreceptors to adjacent second-order neurons have been shown to cause congenital stationary night blindness (CSNB). Sequence alterations in CACNA1F lead to the incomplete type of CSNB (CSNB2), which can be distinguished by standard electroretinography (ERG). CSNB2 is associated with a reduced rod b-wave, a substantially reduced cone a-wave, and a reduced 30-Hz flicker ERG response. CACNA1F encodes the alpha 1-subunit of an L-type Ca2+ channel (Cav1.4 alpha ), which is specific to photoreceptors and is present at high density in the synaptic terminals. Ten of our patients with CSNB2 showed no mutation in CACNA1F. To identify the disease-causing mutations, we used a candidate-gene approach. CABP4, a member of the calcium-binding protein (CABP) family, is located in photoreceptor synaptic terminals and is directly associated with the C-terminal domain of the Cav1.4 alpha . Mice lacking either Cabp4 or Cav1.4 alpha display a CSNB2-like phenotype. Here, we report for the first time that mutations in CABP4 lead to autosomal recessive CSNB. Our studies revealed homozygous and compound heterozygous mutations in two families. We also show that these mutations reduce the transcript levels to 30%-40% of those in controls. This suggests that the reduced amount of CABP4 is the reason for the signaling defect in these patients.

The ICF syndrome (immunodeficiency, centromeric instability, facial anomalies) (OMIM#242860) is a rare autosomal, recessively inherited disorder. Another rare condition, ischiadic hypoplasia, renal dysgenesis, immunodeficiency, and polydactyly (IHRDIP, OMIM#243340), displays features that resemble those of the ICF syndrome. Due to the overlapping symptoms in both syndromes, we asked whether a shared underlying molecular defect exists. Two patients, each with the clinical characteristics of one of these syndromes, were subjected to conventional cytogenetic analysis and the determination of the methylation state of satellite II DNA. We found that both displayed the two hallmark features of the ICF syndrome, namely hypomethylation and centromeric instability of chromosomes 1 and 16. Therefore, we reclassified the patient previously diagnosed with the IHRDIP syndrome as an ICF patient. Since the majority of ICF patients are carriers of mutations in the methytransferase gene DNMT3B, we determined the sequence of its coding, splice site, and putative promoter region and analyzed its transcripts in both patients, without detecting any alterations. Similarly, the coding region of two DNMT3B-interacting proteins, SUMO-1 and UBC9, did not reveal mutations. With this study, the published number of patients that lack mutations in DNMT3B coding region increases to almost 40% of all ICF patients reported. It is, therefore, implied that a significant subset of ICF patients will have a yet unknown, alternative alteration, which may include the involvement of DNMT3B-interacting factors or aberrations of an independent pathway.

Purpose Biochemical and genetic analyses established a contribution of lipid metabolism to AMD pathology. Paraoxonase 1 (PON1) is an antioxidative protein involved in high density lipoprotein (HDL) function and was found to be associated with AMD. Here, we used Pon1(-/-) mice to study the influence of PON1 on retinal physiology and to reveal the potential impact of PON1 on AMD etiology. Methods Laser capture microdissection served to isolate single retinal layers. Retinal function was assessed by ERG. Retinal and RPE morphology were monitored by fundus imaging, fluorescein angiography, light and transmission electron microscopy, and immunofluorescence microscopy. Levels of mRNA and composition of phospholipid species were determined by real-time PCR and LC-MS, respectively. Results Adult (8 weeks old) Pon1(-/-) mice displayed normal retinal function and morphology, but their retinas contained reduced amounts of lysophosphatidylcholines (LPCs) compared to controls. Aged (12 months old) Pon1(-/-) animals did not show any morphologic or molecular signs of photoreceptor or RPE degeneration, or of accelerated aging. Photoreceptors of Pon1(-/-) and control mice were similarly susceptible to light damage. Conclusions Results indicated that PON1 is not essential for normal development, function, ageing, and the defense against light damage of the mouse retina. Reduced levels of LPCs in eyes of Pon1(-/-) mice may reflect a decreased activity of phospholipase A2 or altered antioxidative activity in aged eyes.

Human Molecular Genetics, 22 (16), ISSN:0964-6906, ISSN:1460-2083

Age, 35 (5), ISSN:0161-9152, ISSN:1574-4647

We have studied a germinal revertant of the Mutator (Mu3)-induced mutation (Adh1-3F1124) of the maize alcohol dehydrogenase 1 gene (adh1). Transposon Mu3 was inserted at the TATA box of the promoter. The excision of Mu3 caused a complex, multibreakpoint DNA rearrangement with deletion, inverted duplication, and inversions affecting 430 nucleotides in the promoter region. These changes led to an unusual pattern of adh1 gene expression: increased levels of enzyme activity in one organ, decreased levels in another, and almost unchanged levels in a third organ. The evolutionary impact of transposon-induced promoter scrambling on generation of allelic diversity is discussed. We present a fragmentation model to help explain how transposon excision could induce multiple breakpoint aberrations without involving a homologous chromosome.

Item does not contain fulltext Wagner syndrome (WS) is an autosomal dominant vitreoretinopathy affecting various ocular features and is caused by mutations in the canonical splice sites of the VCAN gene, which encodes the large chondroitin sulfate proteoglycan, versican. We report the identification of novel splice acceptor and donor-site mutations (c.4004-1G>C and c.9265+2T>A) in two large WS families from France and the United Kingdom. To characterize their pathogenic mechanisms we performed qRT-PCR experiments on RNA from patient-derived tissues (venous blood and skin fibroblasts). We also analyzed RNA from the original Swiss family reported by Wagner (who has the previously reported c.9265+1G>A mutation). All three mutations resulted in a quantitative increase of transcript variants lacking exons 7 and/or 8. However, the magnitude of the increase varied between tissues and mutations. We discuss altered balance of VCAN splice variants in combination with reduction in glycosaminoglycan protein modifications as possible pathogenic mechanisms.

Objective Factors influencing the outcome of anti-VEGF treatment in neovascular AMD are still investigated. We analyzed the impact of a loading phase, the significance of an initial response for the long-term and the effect of the CFH polymorphism (p.His402Tyr) on treatment outcome. Methods Patients treated with ranibizumab for neovascular AMD were analyzed over a period of 24 months by assessing effects of loading phase, initial response and genotype of CFH rs1061170 (c.1204C>T, p.His402Tyr). Results 204 eyes were included. A change of +5.0 [−1;+11] letters and +1.5 [−5.5;+9.5] was observed with a median of 4 [3]; [7] and 10 [7]; [14] ranibizumab injections during 12 and 24 months, respectively. Loading phase was no significant predictor for treatment as VA outcome in eyes with and without loading phase was similar (p = 0.846 and p = 0.729) at 12 and 24 months. In contrast, initial response was a significant predictor for improving vision of 5 or more letters at 12 (p = 0.001; OR = 6.75) and 24 months (p = 0.01; OR = 4.66). Furthermore, the CT genotype at CFH rs1061170 was identified as a significant predictor for a favorable VA outcome at 12 and 24 months (OR = 6.75, p = 0.001 and OR = 4.66, p = 0.01). Conclusions Our data suggest that clinical decisions regarding treatment may be guided by observing patients’ initial response as well as their genotype of SNP rs1061170, while the criterion of loading phase may not bear the customary value., PLoS ONE, 7 (7), ISSN:1932-6203

Neovascular age-related macular degeneration (AMD) resulting in decreased central vision severely impairs affected individuals. Current standard treatment is an intravitreal anti-VEGF therapy (ranibizumab), but responses to treatment show large variability. Genetic factors that influence AMD and that affect the outcome of ranibizumab treatment were sought within a sample of Swiss patients.Changes in visual acuity (VA) after initiation of anti-VEGF treatment were observed during 12 months, and percentiles of VA were calculated. Genotypes of polymorphisms in known AMD susceptibility loci (CFH, CFB, HTRA1, AMRS2, and VEGFA) as well as not yet reported AMD-associated genes (KDR, LRP5, and FZD4) were determined, and their frequencies were compared.Of the 309 eyes included in the study, 243 completed VA assessment. On average, 3.9 ±2.6 ranibizumab injections were administered. Based on the change in visual acuity, two responder groups were established: 63 eyes were assigned to the poor responders (≤25th percentile) and 63 eyes to the good responders (≥75th percentile). Individuals with genotype CC of p.Y402H in CFH had a decreased chance of positive treatment outcome compared with those with the CT and TT genotypes (P = 0.005 and P = 0.006). In this study, the genotype combination of AG at CFH with CT at FZD4 (SNP rs10898563) promised an increased chance of positive treatment outcome (P = 0.004). Furthermore, the association with the known genetic susceptibility loci CFH, HTRA1, and AMRS2 were confirmed, and a risk-conferring polymorphism in one new locus, LRP5, was identified.Genetic predisposition may account for the variability in response to anti-VEGF treatment.

PURPOSE. Knowledge of genetic factors predisposing to age-related cataract is very limited. The aim of this study was to identify DNA sequences that either lead to or predispose for this disease. METHODS. The candidate gene SLC16A12, which encodes a solute carrier of the monocarboxylate transporter family, was sequenced in 484 patients with cataract (134 with juvenile cataract, 350 with age-related cataract) and 190 control subjects. Expression studies included luciferase reporter assay and RT-PCR experiments. RESULTS. One patient with age-related cataract showed a novel heterozygous mutation (c.-17A>G) in the 5'untranslated region (5'UTR). This mutation is in cis with the minor G-allele of the single nucleotide polymorphism (SNP) rs3740030 (c.-42T/G), also within the 5'UTR. Using a luciferase reporter assay system, a construct with the patient's haplotype caused a significant upregulation of luciferase activity. In comparison, the SNP G-allele alone promoted less activity, but that amount was still significantly higher than the amount of the common T-allele. Analysis of SLC16A12 transcripts in surrogate tissue demonstrated striking allele-specific differences causing 5'UTR heterogeneity with respect to sequence and quantity. These differences in gene expression were mirrored in an allele-specific predisposition to age-related cataract, as determined in a Swiss population (odds ratio approximately 2.2; confidence intervals, 1.23-4.3). CONCLUSIONS. The monocarboxylate transporter SLC16A12 may contribute to age-related cataract. Sequences within the 5'UTR modulate translational efficiency with pathogenic consequences.

Contains fulltext : 89365.pdf (Publisher’s version ) (Closed access) PURPOSE: The majority of patients with X chromosome-linked retinitis pigmentosa (XlRP) carry mutations in the RPGR gene. The authors studied whether patients with RPGR mutations show additional splice defects that may interfere with RPGR properties. METHODS: Patient-derived cell lines with RPGR mutations were raised in suspension. To verify mutations, direct sequencing of PCR products was performed. Patient-specific alterations in RPGR splicing were analyzed by RT-PCR and confirmed by sequencing. Tissue-specific expression levels of RPGR splice variants were quantified by real-time PCR using pools of different human donor tissues. RESULTS: The authors analyzed the splicing of RPGR in seven RP patient-derived lymphoblastoid cell lines carrying hemizygous RPGR mutations. In three patient cell lines, they identified and characterized splice defects that were present in addition to a mutation. These splice defects were likely to interfere with normal RPGR properties. Furthermore, they identified four novel RPGR transcripts, either containing a new exon termed 11a or skipping the constitutive exons 12, 14, and 15. Novel and known RPGR isoforms were found to be differentially regulated in several human tissues. In human retina, approximately 10% of RPGR transcripts are alternatively spliced between exons 9 and 15. CONCLUSIONS: These findings show that splicing of RPGR is precisely regulated in a tissue-dependent fashion and suggest that mutations in RPGR frequently interfere with the expression of alternative transcript isoforms. These results implicate the importance of RPGR transcript analysis in patients with RP. The authors further discuss RPGR splicing as a modifier of different disease phenotypes described in patients with XlRP. 01 maart 2010

CD31(+)CD45RA(+)RO(-) lymphocytes contain high numbers of T cell receptor circle (TREC)-bearing T cells; however, the correlation between CD31(+)CD4(+) lymphocytes and TREC during aging and under lymphopenic conditions has not yet been sufficiently investigated. We analyzed TREC, telomere length and telomerase activity within sorted CD31(+) and CD31(-) CD4(+) lymphocytes in healthy individuals from birth to old age. Sorted CD31(+)CD45RA(+)RO(-) naive CD4(+) lymphocytes contained high TREC numbers, whereas CD31(+)CD45RA(-)RO(+) cells (comprising < or =5% of CD4(+) cells during aging) did not contain TREC. CD31(+) overall CD4(+) cells remained TREC rich despite an age-related tenfold reduction from neonatal (100 : 1000) to old age (10 : 1000). Besides a high TREC content, CD31(+)CD45RA(+)RO(-)CD4(+) cells exhibited significantly longer telomeres and higher telomerase activity than CD31(-)CD45RA(+)RO(-)CD4(+) cells, suggesting that CD31(+)CD45RA(+)RO(-)CD4(+) cells represent a distinct population of naive T cells with particularly low replicative history. To analyze the value of CD31 in lymphopenic conditions, we investigated six children after allogeneic hematopoietic stem cell transplantation (HSCT). Reemerging overall CD4(+) as well as naive CD45RA(+)RO(-)CD4(+) cells predominantly expressed CD31 and correlated well with the recurrence of TREC 5-12 months after HSCT. Irrespective of limitations in the elderly, CD31 is an appropriate marker to monitor TREC-rich lymphocytes essentially in lymphopenic children after HSCT.

Deficiencies of vitamin A and iodine are common in many developing countries. Vitamin A deficiency (VAD) may adversely affect thyroid metabolism. The study aim was to investigate the effects of concurrent vitamin A and iodine deficiencies on the thyroid-pituitary axis in rats.Weanling rats (n = 56) were fed diets deficient in vitamin A (VAD group), iodine (ID group), vitamin A and iodine (VAD + ID group), or sufficient in both vitamin A and iodine (control) for 30 days in a pair-fed design. Serum retinol (SR), thyroid hormones (FT(4), TT(4), FT(3), and TT(3)), serum thyrotropin (TSH), pituitary TSHbeta mRNA expression levels, and thyroid weights were determined at the end of the depletion period.Compared to the control and ID groups, SR concentrations were about 35% lower in the VAD and VAD + ID groups (p0.001), indicating moderate VA deficiency. Comparing the VAD and control groups, there were no significant differences in TSH, TSHbeta mRNA, thyroid weight, or thyroid hormone levels. Compared to the control group, serum TSH, TSHbeta mRNA, and thyroid weight were higher (p0.05), and FT4 and TT4 were lower (p0.001), in the VAD + ID and ID groups. Compared to the ID group, TSH, TSHbeta mRNA, and thyroid weight were higher (p0.01) and FT(4) and TT(4) were lower (p0.001) in the VAD + ID group. There were no significant differences in TT3 or FT3 concentrations among groups.Moderate VAD alone has no measurable effect on the pituitary-thyroid axis. Concurrent ID and VAD produce more severe primary hypothyroidism than ID alone.

The aim of the present study was to determine the genetic defect in Wagner syndrome, a rare disorder belonging to the group of hereditary vitreoretinal degenerations. This disease has been genetically mapped to chromosome 5q14.3.Molecular analysis was performed in the progeny of the original pedigree described by Wagner in 1938. We searched for pathogenic mutations and their effects in two candidate genes, CSPG2 and EDIL3, which locate to the critical chromosomal interval. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to investigate potential splice defects of CSPG2 transcripts.While no alterations were detected in the exons of EDIL3, several changes were identified in the CSPG2 gene. Only one of the novel changes, a heterozygous G to A substitution of the first nucleotide in intron 8, cosegregates with the disease phenotype. This change disrupts the highly conserved splice donor sequence. In blood cells of an index patient, we found CSPG2 transcripts with normally spliced exon 8/9 junction but also two additional CSPG2 transcripts, which were not detected in the control. One lacks the entire exon 8, while the other is missing only the last 21 bp of exon 8.CSPG2 encodes versican, a large proteoglycan, which is an extracellular matrix component of the human vitreous and participates in the formation of the vitreous gel. The splice site mutation described here may lead to a complete lack of exon 8 in CSPG2 transcripts, which shortens the predicted protein by 1754 amino acids and leads to severe reduction of glycosaminoglycan attachment sites.

SLC16A12 encodes an orphan member of the monocarboxylate transporter family, MCT12. A nonsense mutation in SLC16A12 (c.643CT; p.Q215X) causes juvenile cataract with a dominant inheritance pattern. In the present study, in vitro and in vivo experimental models were used to gain insight into how the SLC16A12 (c.643CT) mutation leads to cataract formation.MCT12 peptide antibodies were generated and used to examine the expression of MCT12 in the lens using immuno-confocal microscopy. To determine whether loss of Slc16a12 resulted in cataract formation, a Slc16a12 hypomorphic rat generated by transposon insertional mutagenesis was characterized using RT-PCR, slit lamp microscopy and histologic methods. Exogenous expression of MCT12 and MCT12:214Δ, a mimic of the mutant allele, were used to assess protein expression and trafficking.MCT12 protein was detected in the lens epithelium and secondary fiber cells at postnatal day 1. In the Slc16a12(TKO) rat, complete loss of MCT12 did not result in any detectable ocular phenotype. Exogenous expression of MCT12-GFP and MCT12:214Δ-GFP revealed that the full-length protein was trafficked to the plasma membrane (PM), whereas the truncated protein was retained in the endoplasmic reticulum (ER). When both MCT12 and MCT12:214Δ were coexpressed, to mimic the heterozygous patient genotype, the truncated protein was retained in the ER whereas full-length MCT12 was trafficked to the PM. Furthermore, MCT12 was identified as another MCT isoform that requires CD147 for trafficking to the cell surface.These data support a model whereby the SLC16A12 (c.643CT) mutation causes juvenile cataract by a defect in protein trafficking rather than by haploinsufficiency.

Objective— The mechanisms responsible for the age-related increase in the incidence of calcific aortic valve stenosis (CAS) are unclear but may include telomere-driven cellular senescence. Because telomere length varies widely among individuals of the same age, we hypothesized that patients with shorter telomeres would be prone to develop CAS late in life. Methods and Results— Mean telomere length was measured in leukocytes from a cohort of 193 patients ≥70 years of age with and without CAS. Pilot experiments performed in 30 patients with CAS and controls pair-matched for age, sex, and presence or absence of coronary disease demonstrated significantly shorter telomeres in the CAS group both by Southern blot hybridization (5.75±0.55 kbp versus 6.27±0.7 kbp, P =0.0023) and by a quantitative polymerase chain reaction-based technique (relative telomere length 0.88±0.19 versus 1.0±0.19, P =0.01). This finding was then confirmed in the whole cohort (CAS n=64, controls n=129, relative telomere length=0.86±0.16 versus 0.94±0.12, P =0.0003). Both groups were comparable for potential confounding characteristics. Subgroup analysis according to the presence or absence of coronary disease demonstrated no association of this disorder with telomere length. Conclusions— In the elderly, calcific aortic stenosis, but not coronary disease, is associated with shorter leukocyte telomere length.

Due to an oversight, the manuscript ‘‘A New and a Reclassified ICF Patient Without Mutations in DNMT3B and its Interacting Proteins SUMO-1 and UBC9’’ by KloeckenerGruissem B, Betts DR, Zankl A, Berger W, Gungor T in Am J Med Genet A, 2005 July 1;136:31–37 had been submitted and subsequently published with an incorrect affiliation for one of the contributors. The correct affiliation for Andreas Zankl should read: ‘‘Institute of Medical Genetics, University of Zurich, Switzerland.’’ Dr. Zankl regrets the error.

A new, unstable, organ-specific Adh1 mutant was isolated from a Robertson's mutator line by germinating kernels under partial anaerobic conditions. Families of kernels which showed segregation of a conditional anaerobic lethal phenotype were identified. One mutant, Adh1-3F1124, was shown to express approximately 6% normal levels of ADH1 in seed and anaerobically treated seedlings but expresses normal levels of ADH1 in pollen, the male gametophyte. The ADH1 polypeptide encoded by the mutant allele was found to be indistinguishable from that encoded by the Adh1-3F progenitor but its message levels were lower in seed and seedlings. Robertson's mutator lines are known to carry Mu transposons that cause increased mutation rates. Genomic Southern analysis of Adh1-3F1124 and Adh1-3F showed the presence of a 1.85 kbp insertion at the 5′ region of Adh1. Comparison of the DNA sequences revealed that a Mu1-like element was inserted 31 bp 5′ from the transcriptional start site of Adh1-3F1124gene. The insertion of the Mu element creates an additional TATA box by duplicating the 9 bp genomic sequence—ATATAAATC—at the site of insertion. Consequently, there are two potentially functional TATA sequences, separated by the 1.85 kbp Mu element, 5′ to the transcriptional start site. It is not yet understood how such an arrangement alters the organspecific expression of Adh1.

The nuclear PET122 gene from S. cerevisiae is necessary for translation of a single mitochondrial mRNA that encodes subunit III of cytochrome c oxidase. We report here the cloning and nucleotide sequence of PET122, and properties of the predicted protein product, which consists of 242 residues. Analysis of PET122-lacZ translational fusions confirms that the PET122 coding region is translated in vivo and indicates that the PET122 protein product is targeted to mitochondria. A 117 residue domain located in the carboxy-terminal half of the PET122 protein, at least part of which is shown by characterization of mutants to be critical for PET122 function, exhibits 24% identity and 59% similarity to a portion of the catalytic domain of E. coli alanyl-tRNA synthetase. However, pet122 mutants are not defective in mitochondrial translation per se, as would be expected if PET122 encoded a tRNA synthetase. Instead, the PET122 protein may carry out one or more activities in common with tRNA synthetases, such as binding of ATP or RNA.

Fourteen nuclear complementation groups of mutants that specifically affect the three mitochondrially-encoded subunits of yeast cytochrome c oxidase have been characterized. Genes represented by these complementation groups are not required for mitochondrial transcription, transcript processing, or translation per se but are required for the expression of one of the three genes — COX1, COX2, or COX3 — which encode the cytochrome c oxicase subunits I, II, or III, respectively. Five of these genes affect the biogenesis of cytochrome c oxidase subunit I, 3 affect the biogenesis of subunit II, 3 affect the biogenesis of subunit III and 3 affect the biogenesis of both cytochrome c oxidase subunit I and cytochrome b, the product of COB. Among the 5 complementation groups of mutants that affect the expression of COX1, 2 lack COX1 transcripts, 1 produces incompletely processed COX1 transcripts, and 2 contain normal levels of normal-sized COX1 transcripts. In contrast, all 3 complementation groups which affect the expression of COX2 and all 3 complementation groups which affect the expression of COX3 exhibit no, or little, detectable difference with respect to the wild type pattern of transcripts. The 3 complementation groups which affect the expression of both COX1 and COB all have aberrant COX1 and COB transcript patterns. These findings indicate that multiple trans-acting nuclear genes are required for specific expression of each COX gene encoded on mitochondrial DNA and suggest that their products act at different steps in the expression of these mitochondrial genes.