Franck Sturtz

The 50th anniversary of the discovery of trisomy 21: the past, present, and future of research and treatment of Down syndrome.

Trisomy 21 or Down syndrome is a chromosomal disorder resulting from the presence of all or part of an extra Chromosome 21. It is a common birth defect, the most frequent and most recognizable form of mental retardation, appearing in about 1 of every 700 newborns. Although the syndrome had been described thousands of years before, it was named after John Langdon Down who reported its clinical description in 1866. The suspected association of Down syndrome with a chromosomal abnormality was confirmed by Lejeune et al. in 1959. Fifty years after the discovery of the origin of Down syndrome, the term “mongolism” is still inappropriately used; persons with Down syndrome are still institutionalized. Health problems associated with that syndrome often receive no or little medical care, and many patients still die prematurely in infancy or early adulthood. Nevertheless, working against this negative reality, community-based associations have lobbied for medical care and research to support persons with Down syndrome. Different Trisomy 21 research groups have already identified candidate genes that are potentially involved in the formation of specific Down syndrome features. These advances in turn may help to develop targeted medical treatments for persons with Trisomy 21. A review on those achievements is discussed.

Genotype-Phenotype Correlations in Charcot-Marie-Tooth Disease Type 2 Caused by Mitofusin 2 Mutations

BACKGROUND Mutations in the gene encoding mitofusin 2 (MFN2) cause Charcot-Marie-Tooth disease type 2 (CMT2), with heterogeneity concerning severity and associated clinical features. OBJECTIVE To describe MFN2 mutations and associated phenotypes in patients with hereditary motor and sensory neuropathy (HMSN). DESIGN Direct sequencing of the MFN2 gene and clinical investigations of patients with MFN2 mutations. SETTING Molecular genetics laboratory of a university hospital and the Limoges National Referral Center for Rare Peripheral Neuropathies. PATIENTS One hundred fifty index patients with HMSN and a median motor nerve conduction velocity of 25 m/s or greater and without mutations in the genes encoding connexin 32 and myelin protein zero. MAIN OUTCOME MEASURES Results of genetic analyses and phenotypic observations. RESULTS Twenty different missense mutations were identified in 20 index patients. Mutation frequency was 19 of 107 (17.8%) in patients with CMT2 and 1 of 43 (2.3%) in patients with a median motor nerve conduction velocity less than 38 m/s. Four patients had proven de novo mutations, 8 families had autosomal dominant inheritance, and 3 had autosomal recessive inheritance. The remaining 5 patients were sporadic cases with heterozygous mutations. Phenotypes varied from mild forms to early-onset severe forms. Additional features were encountered in 8 patients (32%). Six patients underwent sural nerve biopsy: electronic microscopy showed prominent mitochondrial abnormalities on longitudinal sections. CONCLUSIONS MFN2 mutations are a frequent cause of CMT2, with variable severity and either dominant or recessive inheritance. MFN2 gene testing must be a first-line analysis in axonal HMSN irrespective of the mode of inheritance or the severity of the peripheral neuropathy.

High metabolic level in patients with familial amyotrophic lateral sclerosis

An abnormally elevated level of resting energy expenditure (REE, measured by indirect calorimetry) has been reported in a subset of patients with sporadic amyotrophic lateral sclerosis (SALS). Hypermetabolism (measured REE/calculated REE (cREE)≥1.1, or 110%) has also been observed in transgenic mice harbouring ALS-causing mutations in the SOD1 gene. By contrast, the REE of patients with familial amyotrophic lateral sclerosis (FALS) has never been assessed. Our objective was to evaluate the metabolic and nutritional parameters of FALS patients and to compare them with those of SALS patients, and search for correlations with clinical parameters. Eleven patients with FALS (from 10 different families, none carrying a SOD1 mutation) were evaluated by indirect calorimetry in our centre. As a control group, we used a sample of 33 patients with SALS, matched for age and sex with the FALS patients. 11/11 (100%) patients with FALS were hypermetabolic, compared to 17/33 (52%) patients with SALS (p=0.009). Measured REE (mREE) and mREE/cREE (metabolic level) were significantly higher in FALS patients than in SALS patients (p=0.03 and p=0.0008, respectively). No correlation was found between metabolic measures and neurological or respiratory parameters. In conclusion, hypermetabolism appears to be a common feature of subjects with FALS, suggesting that this impairment of energy homeostasis may be genetically driven. The high metabolic level of FALS patients should be taken into account for their nutritional management (need for a high-energy diet to prevent malnutrition).

Severe early-onset axonal neuropathy with homozygous and compound heterozygous MFN2 mutations.

Objective: Severe early-onset axonal neuropathy (SEOAN) is a heterogeneous phenotype first delineated by Ouvrier et al., characterized by progressive axonal degeneration with gait problems often progressing to wheelchair requirement and later respiratory involvement. Most cases are sporadic single cases. Some have heterozygous mitofusin 2 (MFN2) mutations, many of which are de novo dominant mutations. The aim of this study was to investigate the mode of inheritance in three individuals with severe early-onset axonal neuropathy and homozygous or compound heterozygous MFN2 mutations. Methods: The clinical and molecular findings in the parents of three individuals with SEOAN with homozygous or compound heterozygous MFN2 mutations were examined. Results: All parents were asymptomatic or mildly symptomatic with some signs of peripheral neuropathy indicating a minimal phenotype. Two had hearing problems. All parents carried the relevant single base (heterozygous) MFN2 variations. Conclusion: Severe early-onset axonal neuropathy due to MFN2 mutations can present as an apparently recessively inherited neuropathy but the minimal phenotype in the parents suggests a semi-dominant mechanism.

Gene expression of HIF-1α and XRCC4 measured in human samples by real-time RT-PCR using the sigmoidal curve-fitting method

Quantitative reverse transcription PCR (RT-PCR) has become an important tool for studying functional gene expression. However the most often used cycle threshold (CT)-based method, primarily related to the required amplification efficiency determination via serial dilution, can call into question the level of quantitative reliability and accuracy that can be achieved, in addition to the impracticalities inherent to CT-based methodologies. In this study, an alternative method, named the sigmoidal curve-fitting (SCF) method, was compared with the classic CT method for two target genes (XRCC4 and HIF-1alpha) and a reference gene (HPRT). The PCR conditions were optimized for each gene on a LightCycler apparatus. Fluorescence data were fitted to a four-parametric sigmoidal function, and the initial messenger RNA (mRNA) copy number was determined by a theoretical fluorescence (F0) value calculated from each fitting curve. The relative expression of the target gene versus that of the reference gene was calculated using an equation based upon these F0 values. The results show that the F0 value had a good linearity with the initial number of target genes between 10(7) and 10(1) copies. The reproducibility tests showed that the variations of initial target quantity were well reflected by F0 values. Relative expression of target gene calculated by the SCF method and by the CT method showed similar results. In our hands, the SCF method gave reliable results and a more precise error description of quantitative RT-PCR.

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.

Effect of Leucovorin (Folinic Acid) on the Developmental Quotient of Children with Down's Syndrome (Trisomy 21) and Influence of Thyroid Status

Background Seven genes involved in folate metabolism are located on chromosome 21. Previous studies have shown that folate deficiency may contribute to mental retardation in Downs syndrome (DS). Methodology We investigated the effect of oral folate supplementation (daily dose of 1.0±0.3 mg/kg) on cognitive functions in DS children, aged from 3 to 30 months. They received 1 mg/kg leucovorin or placebo daily, for 12 months, in a single-centre, randomised, double-blind study. Folinic acid (leucovorin, LV) was preferred to folic acid as its bioavailability is higher. The developmental age (DA) of the patients was assessed on the Brunet-Lezine scale, from baseline to the end of treatment. Results The intent-to-treat analysis (113 patients) did not show a positive effect of leucovorin treatment. However, it identified important factors influencing treatment effect, such as age, sex, and concomitant treatments, including thyroid treatment in particular. A per protocol analysis was carried out on patients evaluated by the same examiner at the beginning and end of the treatment period. This analysis of 87 patients (43 LV-treated vs. 44 patients on placebo) revealed a positive effect of leucovorin on developmental age (DA). DA was 53.1% the normal value with leucovorin and only 44.1% with placebo (p<0.05). This positive effect of leucovorin was particularly strong in patients receiving concomitant thyroxin treatment (59.5% vs. 41.8%, p<0.05). No adverse event related to leucovorin was observed. Conclusion These results suggest that leucovorin improves the psychomotor development of children with Downs syndrome, at least in some subgroups of the DS population, particularly those on thyroxin treatment. Trial Registration ClinicalTrials.gov, NCT00294593

Involvement of Human Herpesvirus-6 Variant B in Classic Hodgkin's Lymphoma via DR7 Oncoprotein

Purpose: Hodgkins lymphoma (HL) is associated with the presence of EBV in Reed-Sternberg (RS) cells in ∼40% of cases. Here, we studied the presence of human herpesvirus type 6 (HHV-6) variant B in RS cells of HL patients and correlated results with clinical parameters. We then examined the implication of HHV-6 DR7B protein in cell deregulation. Experimental Design: HHV-6 DR7B protein was produced in a Semliki Forest virus system. Polyclonal antibodies were then generated and used for immunochemical HHV-6 localization in HL biopsies. Binding between DR7B and p53 was studied using a double-hybrid system. Transactivation of NFκB was observed after transient transfection using reporter gene assays. We looked for Id2 factor expression after stable transfection of the BJAB cell line by reverse transcription-PCR and Western blot analysis. Results: HHV-6 was more common in nodular sclerosis subtype HL, and DR7B oncoprotein was detected in RS cells for 73.7% of EBV-negative patients. Colocalization of EBV and HHV-6 was observed in RS cells of doubly infected patients. DR7B protein bound to human p53 protein. p105-p50/p65 mRNA expression and activation of the NFκB complex were increased when DR7B was expressed. Stable expression of DR7B exhibited a strong and uniform expression of Id2. A slightly higher percentage of remission was observed in patients with RS cells testing positive for DR7B than in those testing negative. Conclusions: Collectively, these data provide evidence for the implication of a novel agent, HHV-6, in cases of nodular sclerosis HL. Clin Cancer Res; 16(19); 4711–21. ©2010 AACR.

Expression of 5-lipoxygenase (5-LOX) in T lymphocytes.

5‐lipoxygenase (5‐LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5‐LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5‐LOX was amplified by conventional reverse transcription–polymerase chain reaction (RT‐PCR; MOLT4 and Jurkat cells) and by in situ RT‐PCR (T lymphocytes). 5‐LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5‐LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis‐supporting medium. Fluorescence‐activated cell sorter analysis of different T‐lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T‐cell receptor‐αβ and ‐γδ expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C4 and a small amount of leukotriene B4 in response to CD3–CD28 cross‐linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK‐886 and AA‐861. The presence of 5‐LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Diagnostic value of ultrastructural nerve examination in Charcot-Marie-Tooth disease: two CMT 1B cases with pseudo-recessive inheritance

We report two sporadic patients of CMT disease in different consanguineous families. The electrophysiological examination led to the diagnosis of a severe demyelinating neuropathy. The nerve biopsies exhibited numerous outfoldings of the myelin sheaths and onion-bulb proliferations. The consanguinity and the histological findings pointed to a diagnosis of CMT 4B. However, the detection of abnormal and regular widenings between the major dense lines of the myelin lamellae by electron microscopy led us to search for a P0 gene mutation. Two heterozygous mutations of this gene were identified: S63F and N131Y. Different aspects of uncompacted myelin lamellae have been described in some cases of P0 mutations and a few now appear to be quite specific to it. More than 30 genes are implicated in CMT and as mutation search is time- and money-consuming, we believe that in some selected patients ultrastructural examination of nerves, among other criteria, helps orientate the molecular diagnosis of CMT.