Cécile Acquaviva

CDKN2A, CDKN2B, and MTAP gene dosage permits precise characterization of mono‐ and bi‐allelic 9p21 deletions in childhood acute lymphoblastic leukemia

Deletion of the 9p21 chromosomal region is frequently found in childhood acute lymphoblastic leukemia (ALL). The target of these deletions is CDKN2A, a gene encoding both p16INK4a and p14ARF. However, contiguous genes such as CDKN2B, encoding p15INK4b, or MTAP, encoding methylthioadenosine phosphorylase, can be included in the deletions. Gene dosage by use of real‐time PCR has recently been proposed as a promising technical option for the diagnosis of deletions. However, its reliability and its capacity to detect mono‐allelic deletions in tumor samples are controversial. To evaluate the frequency and extent of deletions in 284 children with ALL, we devised a real‐time PCR assay for CDKN2A, CDKN2B exons 1β and 3, and MTAP gene dosage and validated it by comparison with loss‐of‐heterozygosity analysis. We show that, if several controls and adjustments are performed, real‐time PCR can provide a reliable test for mono‐ and bi‐allelic deletions in ALL. We propose a strategy that overcomes the major caveats of such a dosage in tumor samples: aneuploidy and contamination by normal cells. By use of this assay, we found bi‐allelic deletions in 58 and 17% of T‐ and B‐lineage ALL, respectively. Mono‐allelic deletion was observed in about 15% of cases, stressing the importance of their detection in ALL. CDKN2B and/or MTAP co‐deletions were highly variable in both T‐ and B‐lineage ALL, making ALL with 9p21 a rather heterogeneous group. Because proteins encoded by these genes might influence the response to treatment, the prognosis of 9p21‐deleted ALL could vary according to the extent of the deletion.

Genotype-phenotype correlation in primary hyperoxaluria type 1: the p.Gly170Arg AGXT mutation is associated with a better outcome

We sought to ascertain the long-term outcome and genotype-phenotype correlations available for primary hyperoxaluria type 1 in a large retrospective cohort study. We examined the clinical history of 155 patients (129 families primarily from Western Europe, North Africa, or the Middle East) as well as the enzymatic or genetic diagnosis. The median age at first symptom was 4 years, and at diagnosis 7.7 years, at which time 43% had reached end-stage renal disease. Presentations included: (1) early nephrocalcinosis and infantile renal failure, (2) recurrent urolithiasis and progressive renal failure diagnosed during childhood, (3) late onset with occasional stone passage diagnosed in adulthood, (4) diagnosis occurring on post-transplantation recurrence, and (5) family screening. The cumulative patient survival was 95, 86, and 74% at ages 10, 30, and 50 years, respectively, with the cumulative renal survival of 81, 59, 41, and 10% at ages 10, 20, 30, and 50 years, respectively; 72 patients had undergone a total of 97 transplantations. Among the 136 patients with DNA analysis, the most common mutation was p.Gly170Arg (allelic frequency 21.5%), with a median age at end-stage renal disease of 47 years for homozygotes, 35 years for heterozygotes, and 21 years for other mutations. Our results underscore the severe prognosis of primary hyperoxaluria type 1 and the necessity for early diagnosis and treatment, as well as confirm a better prognosis of the p.Gly170Arg mutation.

Single-Nucleotide Polymorphism Phylotyping of Escherichia coli†

ABSTRACT We describe a rapid and easily automated phylogenetic grouping technique based on analysis of bacterial genome single-nucleotide polymorphisms (SNPs). We selected 13 SNPs derived from a complete sequence analysis of 11 essential genes previously used for multilocus sequence typing (MLST) of 30 Escherichia coli strains representing the genetic diversity of the species. The 13 SNPs were localized in five genes, trpA, trpB, putP, icdA, and polB, and were selected to allow recovery of the main phylogenetic groups (groups A, B1, E, D, and B2) and subgroups of the species. In the first step, we validated the SNP approach in silico by extracting SNP data from the complete sequences of the five genes for a panel of 65 pathogenic strains belonging to different E. coli pathovars, which were previously analyzed by MLST. In the second step, we determined these SNPs by dideoxy single-base extension of unlabeled oligonucleotide primers for a collection of 183 commensal and extraintestinal clinical E. coli isolates and compared the SNP phylotyping method to previous well-established typing methods. This SNP phylotyping method proved to be consistent with the other methods for assigning phylogenetic groups to the different E. coli strains. In contrast to the other typing methods, such as multilocus enzyme electrophoresis, ribotyping, or PCR phylotyping using the presence/absence of three genomic DNA fragments, the SNP typing method described here is derived from a solid phylogenetic analysis, and the results obtained by this method are more meaningful. Our results indicate that similar approaches may be used for a wide variety of bacterial species.

Carglumic acid: an additional therapy in the treatment of organic acidurias with hyperammonemia?

BackgroundHyperammonemia in patients with methylmalonic aciduria (MMA) and propionic aciduria (PA) is caused by accumulation of propionyl-CoA which decreases the synthesis of N-acetyl-glutamate, the natural activator of carbamyl phosphate synthetase 1. A treatment approach with carglumic acid, the structural analogue of N-acetyl-glutamate, has been proposed to decrease high ammonia levels encountered in MMA and PA crises.Case presentationWe described two patients (one with MMA and one with PA) with hyperammonemia at diagnosis. Carglumic acid, when associated with standard treatment of organic acidurias, may be helpful in normalizing the ammonia level.ConclusionEven though the usual treatment which decreases toxic metabolites remains the standard, carglumic acid could be helpful in lowering plasma ammonia levels over 400 micromol/L more rapidly.

N219Y, a new frequent mutation among mut(degree) forms of methylmalonic acidemia in Caucasian patients.

Mutations in the MUT locus encoding for the methylmalonyl-CoA mutase (MCM) apoenzyme are responsible for the mut forms of methylmalonic acidemia (MMA). To date, 49 different mutations have been identified in mut MMA. Only two frequent mutations have been reported in the Japanese population and in African-Americans. Here we report a new missense mutation N219Y (731 A→T) which we found in five unrelated families of French and Turkish descent. All the patients exhibited a severe mut° phenotype and three of them were homozygotes for N219Y. Direct involvement of the mutation in the loss of enzyme activity was demonstrated by mutagenesis and transient expression study. Mapping of the mutation onto a three-dimensional model of human MCM constructed by homology with the Propionibacterium shermanii enzyme shows that it lies in a highly conserved secondary structure motif and might suggest impaired folding and/or poor stability compatible with the mut° phenotype. Finally, a 1% N219Y carrier frequency was observed in a French anonymous control population. Thus, N219Y is the first frequent mut mutation to be reported in the Caucasian population.

DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation

Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 μM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 μM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation.

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patients fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency

BACKGROUND Deficiency of mitochondrial trifunctional protein (MTP) is caused by mutations in the HADHA and HADHB genes, which have been mostly delineated at the genomic DNA level and have not been always elucidated. AIM To identify mutations in a French cohort of 52 MTP deficient patients and the susceptibility of mutations generating premature termination codons (PTCs) to the nonsense mRNA mediated decay (NMD). METHODS Mutation screening in fibroblasts was performed at the cDNA level and real-time RT-PCR was used to compare the levels of the different PTC-bearing mRNAs before and after a treatment of fibroblasts by emetine, a translation inhibitor. RESULTS A mutation detection rate of 100% was achieved. A total of 22 novel mutations were identified, including a large-sized genomic deletion in HADHB gene. A high proportion of all identified mutations were non-sense, frameshift and splicing mutations, generating (PTCs), distributed essentially on HADHA coding regions. We could demonstrate that the majority of mutations resulting in PTCs conform to the established rules governing the susceptibility to NMD. CONCLUSION Our results emphasize the value of cDNA analysis in the characterization of HADHA and HADHB mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in MTP defects.

Misdiagnosed Postpartum Psychosis Revealing a late-onset Urea Cycle Disorder

pected. on readmission to the obstetric ward, her treatment included risperidone (2 mg/day), alprazolam (1.5 mg/day), the phenothiazine antipsychotic cyamemazine (62.5 mg/day), and the anticholinergic tropatepine (10 mg/day). Antibiotic treatment was started to treat a possible infection. Unlike in her previous postpartum episodes, Mrs. G’s mental status responded only partially to antipsychotic treatment. A liaison psychiatry consultation was requested. the liaison psychiatrist found a mildly agitated and disoriented young woman who had wet herself and was circling around her bed. the patient was logorrheic, and her speech, which was mostly incoherent, revealed memory impairment. Strikingly, this mild state of confusion fluctuated during the interview, confirming midwives’ reports of hourly changes in the patient’s behavior. Mrs. G anxiously expressed feelings of guilt toward her newborn and the belief that her diagnosis of postpartum psychosis made her less able to take care of her children. She displayed no anger toward her children and no ideas of persecution, infanticide, or suicide. She also expressed the belief that “God talks to humankind through premonitory dreams or providential meetings,” a claim that had previously been interpreted as a mystic delusion. However, it appeared that this view was part of her cultural and religious background, as was later confirmed by her husband, who reported that she had held this belief for a long time and that it was shared by her relatives. overall, Mrs. G was more confused and less delusional than one would have expected in a typical postpartum psychosis. Puzzled by this clinical picture, the psychiatrist reconsidered the diagnosis of postpartum psychosis and extended his examination to assess the differential diagnosis. He found that Mrs. G had chronic headaches and a habitual reluctance to consume meat. His clinical examination revealed little; the patient had a well-tolerated fever, stable blood pressure and pulse, and no signs of severe sepsis. A neurological examination was unremarkable. the psychiatrist ordered immediate blood tests, including ammonia levels. Within an hour, hyperammonemia was confirmed (224 μmol/liter, controls <50 μmol/liter), along with a respiratory alkalosis and a marked inflammatory syndrome. Results of liver function tests, as well as all the other blood tests, were normal. the psychiatrist contacted the internal medicine fellow, who confirmed the need for an immediate multidisciplinary management of a probable late-onset urea cycle disorder. He decided to transfer the patient to the intensive care unit (ICU), despite the reluctance of the obstetrical team, who felt that the patient should instead be in a secure psychiatric facility. In the ICU, an etiologic treatment of urea cycle disorderinduced hyperammonemia was immediately started unMisdiagnosed Postpartum Psychosis Revealing a late-onset Urea Cycle Disorder