Clinical and neuroimaging features of the m.10197G>A mtDNA mutation: New case reports and expansion of the phenotype variability.

Complex I (CI) is the largest component of the mitochondrial respiratory chain (MRC) and it is made up of 7 mitochondrial DNA (mtDNA)-encoded and at least 38 nuclear DNA-encoded subunits. Isolated CI deficiency is the most common single enzyme deficiency in the heterogeneous group of MRC disorders and it is a relatively common etiology of Leigh-like syndrome (LS). With a few exceptions, descriptions of the clinical spectrum of specific mutations in CI are scarce. We here present three unrelated Italian children who harbored the homoplasmic m.10197G>A mutation in MT-ND3 associated with reduced enzyme activity of CI in muscle. Compared with the spectrum of phenotypes seen in 13 previously described families with the same mutation, these children showed some novel clinical features. Two of the boys presented with subacute onset of dystonia, which showed a remitting-relapsing clinical course in one of them. The third boy presented acute symptoms consisting of speech impairment, progressive left-sided hemiparesis, and also vertebral and arterial malformations. In all the children, molecular studies identified a similar mutation load in tissues, and neuroimaging findings were consistent with the features seen in LS. Functional investigations in cultured skin fibroblasts suggested low ATP production in homoplasmic cells. Our results confirm that the m.10197G>A mutation is relevant to these patients' clinical and biochemical phenotypes, which thus expand the array of phenotypes associated with this variant.

Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease.

Gaucher Disease type 1 (GD1) is a lysosomal disorder that affects many systems. Therapy improves the principal manifestations of the condition and, as a consequence, many patients show a modified phenotype which reflects manifestations of their disease that are refractory to treatment. More generally, it is increasingly recognised that information as to how a patient feels and functions [obtained by patient- reported outcome measurements (PROMs)] is critical to any comprehensive evaluation of treatment. A new set of management goals for GD1 in which both trends are reflected is needed. To this end, a modified Delphi procedure among 25 experts was performed. Based on a literature review and with input from patients, 65 potential goals were formulated as statements. Consensus was considered to be reached when ≥75% of the participants agreed to include that specific statement in the management goals. There was agreement on 42 statements. In addition to the traditional goals concerning haematological, visceral and bone manifestations, improvement in quality of life, fatigue and social participation, as well as early detection of long-term complications or associated diseases were included. When applying this set of goals in medical practice, the clinical status of the individual patient should be taken into account.

Novel spondyloepimetaphyseal dysplasia due to UFSP2 gene mutation.

Beukes hip dysplasia is an autosomal dominant disease which has to date been described only in a large South African family of Dutch origin. The patients presented with progressive epiphyseal dysplasia limited to femoral capital epiphysis and their height was not significantly reduced. A unique variant of the ubiquitin-fold modifier 1 (Ufm1)-specific peptidase 2 (UFSP2) gene (c.868T>C) has been reported in all individuals from Beukes family with clinical and radiological diagnosis of Beukes hip dysplasia. Three individuals, propositus, mother, and grandmother, presented with short stature, joint pain, genu vara and a novel spondyloepimetaphyseal dysplasia involving epiphyses predominantly at hips, but also at knees, ankles, wrists and hands, associated with variable degrees of metaphysis and spine involvement. Exome sequencing allowed us to identify the heterozygous variant c.1277A>C of the UFSP2 gene, leading to the missense change p.D426A, in all 3 patients. This mutation is predicted as damaging and, similarly to the mutation originally described in the Beukes family (p. Y290H), directly affects one of the catalytic residues participating in the active site of the protein. This supports the novel notion that loss of catalytic UFSP2 activity, observed in association with different mutants and already experimentally proven in vitro, may have different clinical outcomes.

Development and validation of a quantitative confirmatory method for 30 ß-lactam antibiotics in bovine muscle using liquid chromatography coupled to tandem mass spectrometry.

A method was developed for the confirmatory and quantitative analysis of 30 ß-lactam antibiotic residues in bovine muscle. The method includes 12 penicillins (amoxicillin, ampicillin, cloxacillin, dicloxacillin, mecillinam, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin), 12 cephalosporins (cefacetrile, cefadroxil, cephalexin, cefalonium, cefazolin, cefoperazone, cefotaxime, cefquinome, cefuroxime, desacetyl cephapirin, desfuroylceftiofur cysteine disulfide, desfuroylceftiofur dimer), five carbapenems (biapenem, doripenem, ertapenem, imipenem, meropenem) and faropenem. Samples were extracted using a simple solvent extraction with acetonitrile:water (80:20, v/v) and C18 dispersive solid-phase extraction (d-SPE) clean-up, followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) detection. Chromatography was performed on a reversed phase CSH C18 column, using a binary gradient separation comprising of 0.01% formic acid and 0.2mM ammonium acetate in water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B). The mass spectrometer was operated in the positive electrospray ionisation mode (ESI(+)). Validation was performed following the 2002/657/EC guidelines. Trueness ranged between 69% and 143% and precision ranged between 2.0% and 29.9% under within-laboratory reproducibility conditions. The developed method uses minimal sample preparation and 30 test samples can be analysed by a single analyst in a single day. To the best of our knowledge, this is the first method for carbapenems in foodstuff that does not require derivatisation.

Increased CD1D polymorphism: identification of two novel alleles, CD1D*03 and *04, in individuals from Morocco.

Two novel CD1D alleles were identified in unrelated individuals from Morocco. They differ each from the common CD1D*01 allele by one nucleotide substitution in exon 2 resulting in one amino acid change in the G-ALPHA1-LIKE domain. According to the IMGT unique numbering for G domain, CD1D*03 has one nucleotide transition c136 > t in codon 46, with an arginine-to-cysteine amino acid change (R46 > C) in the D-STRAND, whereas CD1D*04 has one transition c98 > t in codon 33, with a threonine-to-methionine amino acid change (T33 > M) in the C-STRAND. This suggests that CD1D is more polymorphic than previously assumed.

A nationwide survey of PMM2-CDG in Italy: high frequency of a mild neurological variant associated with the L32R mutation.

PMM2-CDG (PMM2 gene mutations) is the most common congenital disorder of N-glycosylation. We conducted a nationwide survey to characterize the frequency, clinical features, glycosylation and genetic correlates in Italian patients with PMM2-CDG. Clinical information was obtained through a questionnaire filled in by the referral physicians including demographics, neurological and systemic features, neuroimaging data and genotype. Glycosylation analyses of serum transferrin were complemented by MALDI-Mass Spectrometry (MALDI-MS). Between 1996 and 2012, data on 37 Italian patients with PMM2-CDG were collected. All the patients with a severe phenotype were unable to walk unaided, 84 % had severe intellectual disability and 81 % microcephaly. Conversely, among 17 mildly affected patients 82 % had independent ambulation, 64 % had borderline to mild intellectual disability and 35 % microcephaly. Epilepsy and stroke-like events did not occur among patients with the mild phenotype. The rate and extent of systemic involvement were more pronounced in severely affected patients. The L32R misfolding mutation of the PMM2 gene occurred in 70 % of the patients with the mild phenotype and was associated with a less severe underglycosylation of serum Tf at MALDI-MS analyses. Despite their different disease severity, all patients had progressive (olivo)ponto-cerebellar atrophy that was the hallmark clinical feature for the diagnosis. A mild neurological phenotype of PMM2-CDG marked by preserved ambulatory ability and autonomy and associated with L32R mutation is particularly frequent in Italy. PMM2-CDG should be considered in patients with even mild developmental disability and/or unexplained progressive cerebellar atrophy.

The effectiveness of domestic cook on inactivation of murine norovirus in experimentally infected Manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with murine norovirus (MNV). METHODS AND RESULTS: A cooking pan was modified to enable electronic temperature probes to be positioned to record both flesh and environment temperature. Manila clams were infected with 10(4) TCID 50% ml(-1) of MNV. The infected whole-in-shell clams, divided into three replicates, were cooked on an electric stove, and groups of nine clams were removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation to ascertain residual viral load. CONCLUSION: Results showed that 10 min of cooking by a traditional domestic method at a temperature close to 100°C, for at least 2 min, can completely devitalize the MNV in infected clams. This is generally the time needed for the majority of valves to open up. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, it is highly recommended to label all lagoon products as 'requiring cooking before consumption', but no specifications are given on how long and at what temperature they should be cooked. Our results can provide the consumer with useful indications on how to cook clams to prevent any risk of foodborne illness.

The shrunken, bright cerebellum: a characteristic MRI finding in congenital disorders of glycosylation type 1a.

SUMMARY: CDG-1a is an early-onset neurodegenerative disease with selective hindbrain involvement and highly variable clinical presentation. We retrospectively reviewed the clinical records and MR imaging studies of 5 children (3 boys and 2 girls aged 12 days to 2 years at presentation) with molecularly confirmed CDG-1a. The cerebellum was hypoplastic at presentation in 4 cases, progressive bulk loss involved the cerebellum and the pons in all cases, and the cerebellar cortex and subcortical white matter were hyperintense on T2-weighted and FLAIR images in all. We conclude that CDG-1a likely results from a combination of cerebellar hypoplasia and atrophy. Cerebellar volume loss with diffuse T2/FLAIR hyperintensity seems to be a peculiar association in the field of cerebellar atrophies, and may be useful to address the differential diagnosis.

Sentinel lymph node biopsy in gastric cancer: possible applications and limits.

AIMS: To explore the feasibility and accuracy of sentinel lymph node (SLN) biopsy in gastric cancer. PATIENTS AND METHODS: Twenty-nine patients with clinical T1 and T2 N0 M0 gastric cancer less than 5 cm in diameter underwent SLN biopsy with the intraoperative Patent blue method. The procedure continued with radical gastrectomy and D2 lymphadenectomy. We investigated all technical aspects of the blue dye technique and determined the accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the SLN technique. RESULTS: SLNs were detected in 28 of 29 patients; the total number of excised SLNs was 45, with a median of two (range 1-3). Seventeen patients had metastatic SLN, with 21 lymph nodes retrieved. Twenty-two patients had SLNs located at the first level. Four patients had SLNs at the second level, one at the first and second levels, and one at the first and third levels. Five patients had false negative SLNs. The ability of SLN biopsy to predict the status of the other lymph nodes was summarised by an accuracy of 75%, a sensitivity of 75%, a specificity of 75%, a positive predictive value of 88%, and a negative predictive value of 55%. CONCLUSIONS: Our study demonstrates that pick-up SLN biopsy in gastric cancer is technically feasible but has very low sensitivity. Regarding the utility of SLN navigation when attempting to detect the nodal basin, the high rate of false negative SLNs and lymph node level jumping warrant further studies with a large accrual before the method can be introduced into daily practice.

Pontocerebellar hypoplasia: clinical, pathologic, and genetic studies.

BACKGROUND: Mutations in genes encoding subunits of the tRNA-splicing endonuclease (TSEN) complex were identified in patients with pontocerebellar hypoplasia 2 (PCH2) and pontocerebellar hypoplasia 4 (PCH4). OBJECTIVE: We report molecular genetic findings in 12 Italian patients with clinical and MRI findings compatible with PCH2 and PCH4. METHODS: We retrospectively selected a cohort of 12 children from 9 Italian families with MRI of hypoplastic pontocerebellar structures and clinical manifestations suggesting either PCH2 or PCH4 and submitted them to direct sequencing of the genes encoding the 4 subunits of the TSEN complex, namely TSEN54, TSEN34, TSEN15, and TSEN2. RESULTS: In a cohort of 12 children, we detected the common p.A307S mutation in TSEN54 in 9/12 available patients from nine unrelated families. We also detected a novel c.1170_1183del (p. V390fs39X) in compound heterozygosity with the common p.A307S in a child with a severe PCH4 phenotype. In another severely affected patient, the second mutant allele was not identified. Two sibs without mutations in the TSEN complex were unlinked to the PCH3 locus. In addition to typical clinical and neuroradiologic features of PCH2, both children were affected by a tubulopathy resembling Bartter syndrome. CONCLUSIONS: We confirm that the common p.A307S mutation in TSEN54 is responsible for most of the patients with a PCH2 phenotype. The presence of a heterozygous in/del variant correlates with a more severe phenotype as PCH4. In addition, we describe a new clinical form of PCH in 2 sibs with clinical and MRI features of PCH2.

Stüve-Wiedemann syndrome: long-term follow-up and genetic heterogeneity.

Stüve-Wiedemann syndrome (SWS, OMIM 601559) is a severe autosomal recessive condition caused by mutations in the leukemia inhibitory receptor (LIFR) gene. The main characteristic features are bowing of the long bones, neonatal respiratory distress, swallowing/sucking difficulties and dysautonomia symptoms including temperature instability often leading to death in the first years of life. We report here four patients with SWS who have survived beyond 36 months of age with no LIFR mutation. These patients have been compared with six unreported SWS survivors carrying null LIFR mutations. We provide evidence of clinical homogeneity of the syndrome in spite of the genetic heterogeneity.

Type II sialidosis: review of the clinical spectrum and identification of a new splicing defect with chitotriosidase assessment in two patients.

Sialidosis is a lysosomal storage disease caused by the deficiency of alpha-N-acetyl neuraminidase-1 (NEU1). Sialidosis is classified into two main clinical variants: Type I, the milder form of the disease, and Type II, which can in turn be subdivided into three forms: congenital, infantile and juvenile. We report herein the clinical, biochemical and molecular characterisation of two patients with Type II sialidosis exhibiting the congenital (P1) and infantile forms (P2). We also review clinical data on the rare Type II forms of sialidosis in the hope of improving understanding of the disorder and facilitating its diagnosis. The genetic characterization of the two patients showed one known [c. 679G > A (p.G227R)] NEU1 missense mutation (detected in P2), and the new c.807 + 1G > A splicing defect (detected in P1), a genetic lesion that is extremely rare in this disease. Interestingly, P2 presented an extremely elevated level of chitotriosidase in plasma. This is the first pathological detection of chitotriosidase in sialidosis patients.

MR imaging findings in 2 cases of late infantile GM1 gangliosidosis.

SUMMARY: Late infantile GM1 gangliosidosis is a rare lysosomal disorder characterized by mental deterioration and progressive spastic, cerebellar, and extrapyramidal signs, without facial dysmorphisms and organomegaly. Neuroimaging findings have been reported in only a few cases. Here we report on predominant globus pallidus MR signal-intensity abnormalities in 2 patients with the late infantile form of GM1 gangliosidosis.

Differential diagnosis and management of focal ground-glass opacities.

Focal pulmonary ground-glass opacities (GGOs) can be associated with bronchioloalveolar carcinoma. The present retrospective study aimed to test the validity of a multistep approach to discriminate malignant from benign localised (focal) GGOs, identifies useful diagnostic features on computed tomography (CT), and suggests appropriate management guidelines. A stepwise approach, including oral antibiotics, follow-up high-resolution CT (HRCT) 40-60 days later and CT-guided core biopsy, was used. All cases with localised GGOs detected since 2001 were reviewed. CT features were described according to a structured scheme. In total, 40 patients were evaluated. Of these, 11 patients were diagnosed with benign GGOs, 19 patients had lung cancer and 10 were undetermined. Nonpolygonal shape, apparent radial growth and clear-cut margins were associated with a malignant histology. The specificity of CT findings was low. Diagnostic accuracy increased after oral antibiotics, follow-up HRCT and percutaneous core biopsy. Overall, 18 patients underwent surgery for lung cancer. In conclusion, malignant ground-glass opacities have a fairly typical appearance, but some benign lesions closely mimic their malignant counterparts. The stepwise approach adopted in the present study increased the diagnostic specificity and reduced time to definitive diagnosis. Segmentectomy might be the ideal resection volume for such tumours.

Molecular and functional characterization of eight novel GAA mutations in Italian infants with Pompe disease.

We characterized 29 unrelated patients presenting with the severe form of Pompe disease (Glycogen Storage Disease Type II, acid maltase deficiency) and identified 26 pathogenic mutations divided over 28 different genotypes. Among the eight new mutations, five were exonic point mutations (c.572A>G, c.1124G>T, c.1202A>G, c.1564C>G and c.1796C>A) leading to codon changes (p.Y191C, p.R375L, p.Q401R, p.P522A and p.S599Y); two were intronic point mutations (c.-32-3C>A and c.1636+5G>C) affecting mRNA processing; one was a single base deletion (c.742delC) generating a truncated protein (p.L248PfsX20). A comprehensive evaluation, based on different methodological approaches, confirmed the detrimental effect of the eight mutations on the protein and its function. Structural alterations potentially induced by the five missense mutations were also predicted through visual inspection of the atomic model of the GAA protein, in terms of both function and spatial orientation of specific residues as well as disturbance generated by amino acid substitutions. Although the remarkable heterogeneity of the mutational spectrum in Pompe disease was already known, our data demonstrate and confirm the power of molecular and functional analysis in predicting the natural course of Pompe disease.

Glycogen storage disease type II: clinical overview.

Glycogen storage disease type II has a broad continuous clinical spectrum in terms of onset, involvement of organs and life expectancy. Infantile onset is the most severe form, presenting with prominent cardiomyopathy, hypotonia, hepatomegaly and death before 12 months of life. Late onset form has onset at any age, lack of severe (or absence of) cardiac involvement, progressive skeletal muscle dysfunction and less dismal short-term prognosis. In addition to muscle and heart involvement, other tissues are affected liver, spleen, endothelium, lung, brain, anterior horns, peripheral nerves. In fact some patients with infantile form have hearing loss, abnormal brain myelination and central fever and some adult patients show aneurysms of brain arteries due to accumulation of glycogen in vessels. As for other treatable lysosomal diseases, the advent of enzyme replacement therapy will change the natural history of this disease and also will increase our knowledge concerning clinical heterogeneity.

Renal allograft immune response is influenced by patient and donor cytokine genotypes.

This study investigated the impact of specific cytokine genotypes on the incidence of acute rejection episodes (ARE), chronic graft dysfunction (CGD), and anti-HLA donor-specific antibody (DS-Ab) production in 86 renal transplant recipients and 70 cadaveric donors. A PCR-SSP method was performed for the analysis of polymorphisms in TNF-alpha, IL-6, TGF-beta, IL-10, and IFN-gamma cytokines. DS-Ab monitoring of sera was performed using a FCXM analysis. Observed cytokine frequencies for patients and donors were not significantly different from the expected frequencies under Hardy-Weinberg equilibrium conditions. The evaluation in recipients revealed a higher frequency of DS-Ab-positive patients among the TNF-alpha high (50.0% vs 25.7%), and for the IL-10 cytokine a greater incidence of ARE-positive patients (35.8% vs 18.2%) with the high + intermediate, compared with the low genotype. The combined effect of these 2 genotypes predisposed to DS-Abs (71.4% vs 25.3%; P = 0.02; odds ratio [OR] = 7.37). As for the TGF-beta1 cytokine, we observed a higher number of CGD-positive patients among high compared with intermediate producers (14.3% vs 0%; P = .050). The analysis of donors revealed a significantly lower incidence of ARE-positive patients among recipients whose donors were carriers of the high IL-6 G/G-genotype compared with the G/C+C/C-genotypes (16.7% vs 41.2%; P = .03), suggesting a protective effect of the G/G genotype on ARE and a predisposing role of donor (-174)allele C. In addition, we noted an association between the IFN-gamma low A/A-genotype and a higher incidence of ARE (42.1% vs 0%; P = .002) and DS-Ab production (47.4% vs 12.5%; P = .02) compared with high producers.

Systemic lupus erythematosus-like disease in a 6-year-old boy with prolidase deficiency.

This report describes the case of a boy with prolidase deficiency who presented with splenomegaly and leg ulcers. Laboratory examination revealed hypergammaglobulinaemia, hyperimmunoglobulinaemia E, increased erythrocyte sedimentation rate, elevated transaminases, positive antinuclear and anti-double-stranded DNA antibodies, and complement consumption. No haematological, renal or articular problems were detected; neutrophil count was normal. The skin lesions were thought to be of vasculitic origin, and a diagnosis of systemic lupus erythematosus was made although the requirements for diagnosis of systemic lupus erythematosus based on American Rheumatism Association criteria were not satisfied. The child was treated with immunosuppressive drugs with worsening of skin lesions before the diagnosis of prolidase deficiency. Prolidase deficiency and systemic lupus erythematosus share a number of common immunological features and at least three patients with prolidase deficiency and immunological and clinical findings fulfilling the diagnostic criteria for systemic lupus erythematosus of the American Rheumatism Association are reported in the literature. Here we review pathogenetic hypothesis linking the metabolic defect to the disturbance in immune function. In particular we discuss the role of highly increased rates of apoptosis and/or abnormal processing of apoptotic keratinocytes in prolidase deficiency and the role of C1q deficiency, which is associated with the failure of normal clearance of apoptotic cells bearing on their surfaces many of the autoantigens involved in systemic lupus erythematosus.