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Diagnosis is essential for the management and treatment of patients with rare diseases. In a group of patients, the genetic study identifies variants of uncertain significance or inconsistent with the phenotype; therefore, it is urgent to develop novel strategies to reach the definitive diagnosis. Herein, we develop the in-house Translational Diagnostics Program (TDP) to validate genetic variants as part of the diagnostic process with the close collaboration of physicians, clinical scientists, and research scientists. The first 7 of 33 consecutive patients for whom exome-based tests were not diagnostic were investigated. The TDP pipeline includes four steps: (i) phenotype assessment, (ii) literature review and prediction of in silico pathogenicity, (iii) experimental functional studies, and (iv) diagnostic decision-making. Re-evaluation of the phenotype and re-analysis of the exome allowed the diagnosis in one patient. In the remaining patients, the studies included either cDNA cloning or PCR-amplified genomic DNA, or the use of patients' fibroblasts. A comparative computational analysis of confocal microscopy images and studies related to the protein function was performed. In five of these six patients, evidence of pathogenicity of the genetic variant was found, which was validated by physicians. The current research demonstrates the feasibility of the TDP to support and resolve intramural medical problems when the clinical significance of the patient variant is unknown or inconsistent with the phenotype.

[Background] Since 1994, over 50 families affected by the episodic ataxia type 1 disease spectrum have been described with mutations in KCNA1, encoding the voltage-gated K+ channel subunit Kv1.1. All of these mutations are either transmitted in an autosomal-dominant mode or found as de novo events., [Methods] A patient presenting with a severe combination of dyskinesia and neonatal epileptic encephalopathy was sequenced by whole-exome sequencing (WES). A candidate variant was tested using cellular assays and patch-clamp recordings., [Results] WES revealed a homozygous variant (p.Val368Leu) in KCNA1, involving a conserved residue in the pore domain, close to the selectivity signature sequence for K+ ions (TVGYG). Functional analysis showed that mutant protein alone failed to produce functional channels in homozygous state, while coexpression with wild-type produced no effects on K+ currents, similar to wild-type protein alone. Treatment with oxcarbazepine, a sodium channel blocker, proved effective in controlling seizures., [Conclusion] This newly identified variant is the first to be reported to act in a recessive mode of inheritance in KCNA1. These findings serve as a cautionary tale for the diagnosis of channelopathies, in which an unreported phenotypic presentation or mode of inheritance for the variant of interest can hinder the identification of causative variants and adequate treatment choice., This study was supported by the Centre for Biomedical Research on Rare Diseases (CIBERER) (ACCI14-759), the URDCat program (PERIS SLT002/16/00174), the Hesperia Foundation and the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia (2017SGR1206) to AP and Instituto de Salud Carlos III (PI14/00581) (cofunded by European Regional Development Fund. ERDF, a way to build Europe) and la Marató de TV3 (345/C/2014) to CC and AP. EV was funded by a grant from the Ministerio de Economia, Industria y Competividad (Juan de la Cierva programme FJCI-2016-28811). SF was funded by the Instituto de Salud Carlos III (Miguel Servet programme CPII16/00016, cofunded by European Social Fund. ESF investing in your future) and MR was funded by CIBERER.

Contains fulltext : 207577.pdf (Publisher’s version ) (Open Access) Brain imaging methods have contributed to shed light on the mechanisms of recovery after early brain insult. The assumption that the unaffected right hemisphere can take over language functions after left perinatal stroke is still under debate. Here, we report how patterns of brain structural and functional reorganization were associated with language outcomes in a group of four-year-old children with left perinatal arterial ischemic stroke (PAIS). Specifically, we gathered specific fine-grained developmental measures of receptive and productive aspects of language as well as standardized measures of cognitive development. We also collected structural neuroimaging data as well as functional activations during a passive listening story-telling fMRI task and a resting state session (rs-fMRI). Children with a left perinatal stroke showed larger lateralization indices of both structural and functional connectivity of the dorsal language pathway towards the right hemisphere that, in turn, were associated with better language outcomes. Importantly, the pattern of structural asymmetry was significantly more right-lateralized in children with a left perinatal brain insult than in a group of matched healthy controls. These results strongly suggest that early lesions of the left dorsal pathway and the associated perisylvian regions can induce the interhemispheric transfer of language functions to right homolog regions. This study provides combined evidence of structural and functional brain reorganization of language networks after early stroke with strong implications for neurobiological models of language development. 18 p.

Neonatal hemochromatosis (NH) has been defined as neonatal liver disorder accompanied by extrahepatic siderosis, and gestational alloimmune liver disease (GALD) is the main cause of NH. We report an atypical case of NH that may have gone underdiagnosed. A male infant was born at term after an uneventful antenatal period. At 7 h of life, he was noted to be tachypneic. Chest X-ray was normal and capillary blood gas analysis showed severe lactic acidosis. An extended blood test showed elevated levels of tyrosine and methionine that, after excluding an inborn error of metabolism, led to the diagnosis of acute liver failure. Hyperferritinemia and elevated transferrin saturation were suggestive of NH. Extrahepatic siderosis on MRI confirmed the diagnosis of NH, so even though the infant was in good general condition, a dose of intravenous immunoglobulin was administered and double volume exchange transfusion was performed. Treatment of a suspected case of GALD and prevention in subsequent gestations is imperative.

Objectives To search for biochemical and molecular markers for the diagnosis of patients and carriers with 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency. Design and methods Organic acids in urine, MHBD activity in fibroblasts, immunoblotting and molecular studies were performed in seven patients. Seven carriers were also studied. Results Under low protein diet or poor feeding all the patients showed only a slightly altered organic acid profile. Measurement of 2-methyl-3-hydroxybutyric acid and tiglylglycine after an isoleucine loading test, failed to demonstrate the carrier status of one patient. However, measurement of 2-ethylhydracrylic acid (EHA) was positive in all the carriers tested. MHBD activity was clearly deficient in males and in one female patient. We identified four missense mutations, two of them were novel. Conclusions Quantification of EHA may be of help for the diagnosis of the heterozygous condition. The carrier females showed the classical biochemical variability of X-linked diseases due to random X-chromosome inactivation.

Brain imaging methods have contributed to shed light on the possible mechanisms of recovery and cortical reorganization after early brain insult. The idea that a functional left hemisphere is crucial for achieving a normalized pattern of language development after left perinatal stroke is still under debate. We report the case of a 3.5-year-old boy born at term with a perinatal ischemic stroke of the left middle cerebral artery, affecting mainly the supramarginal gyrus, superior parietal and insular cortex extending to the precentral and postcentral gyri. Neurocognitive development was assessed at 25 and 42 months of age. Language outcomes were more extensively evaluated at the latter age with measures on receptive vocabulary, phonological whole-word production and linguistic complexity in spontaneous speech. Word learning abilities were assessed using a fast-mapping task to assess immediate and delayed recall of newly mapped words. Functional and structural imaging data as well as a measure of intrinsic connectivity were also acquired. While cognitive, motor and language levels from the Bayley Scales fell within the average range at 25 months, language scores were below at 42 months. Receptive vocabulary fell within normal limits but whole word production was delayed and the child had limited spontaneous speech. Critically, the child showed clear difficulties in both the immediate and delayed recall of the novel words, significantly differing from an age-matched control group. Neuroimaging data revealed spared classical cortical language areas but an affected left dorsal white-matter pathway together with right lateralized functional activations. In the framework of the model for Social Communication and Language Development, these data confirm the important role of the left arcuate fasciculus in understanding and producing morpho-syntactic elements in sentences beyond two word combinations and, most importantly, in learning novel word-referent associations, a building block of language acquisition.

BACKGROUND: Inherited neurometabolic disorders (iNMDs) represent a group of almost seven hundred rare diseases whose common manifestations are clinical neurologic or cognitive symptoms that can appear at any time, in the first months/years of age or even later in adulthood. Early diagnosis and timely treatments are often pivotal for the favorable course of the disease. Thus, the elaboration of new evidence-based recommendations for iNMD diagnosis and management is increasingly requested by health care professionals and patients, even though the methodological quality of existing guidelines is largely unclear. InNerMeD-I-Network is the first European network on iNMDs that was created with the aim of sharing and increasing validated information about diagnosis and management of neurometabolic disorders. One of the goals of the project was to determine the number and the methodological quality of existing guidelines and recommendations for iNMDs. ----- METHODS: We performed a systematic search on PubMed, the National Guideline Clearinghouse (NGC), the Guidelines International Network (G-I-N), the Scottish Intercollegiate Guideline Network (SIGN) and the National Institute for Health and Care Excellence (NICE) to identify all the published guidelines and recommendations for iNMDs from January 2000 to June 2015. The methodological quality of the selected documents was determined using the AGREE II instrument, an appraisal tool composed of 6 domains covering 23 key items. ----- RESULTS: A total of 55 records met the inclusion criteria, 11 % were about groups of disorders, whereas the majority encompassed only one disorder. Lysosomal disorders, and in particular Fabry, Gaucher disease and mucopolysaccharidoses where the most studied. The overall methodological quality of the recommendation was acceptable and increased over time, with 25 % of the identified guidelines strongly recommended by the appraisers, 64 % recommended, and 11 % not recommended. However, heterogeneity in the obtained scores for each domain was observed among documents covering different groups of disorders and some domains like 'stakeholder involvement' and 'applicability' were generally scarcely addressed. ----- CONCLUSIONS: Greater efforts should be devoted to improve the methodological quality of guidelines and recommendations for iNMDs and AGREE II instrument seems advisable for new guideline development. The elaboration of new guidelines encompassing still uncovered disorders is badly needed.

We appreciate the comments of He et al.1 Our response is outlined below. In fact, it had been reported that HSD17B10 is a part of a multigene domain in Xp11.21–p11.22 that escapes X-inactivation.2 Later results by Carrel et al3 showed that this gene is probably subjected to X-inactivation as only one of nine hybrids escapes from it. This observation can not be inferred from Figure 2 of Yang et al,4 while the results are more clarifying in the adapted figure of the letter of He et al.1 To elucidate whether HSD17B10 cDNA doses differed between both sexes, we performed relative quantification (RQ) of wild-type HSD17B10 cDNA alleles in four female and four male controls. The results did not show any significant difference between the doses in both sexes. Therefore, these results are in favour of an X-linked disease that does not escape X-inactivation and are in agreement with the observations of Carrel et al.3 Fibroblasts were obtained from a single biopsy, as it would not have been ethical to perform additional biopsies with the only purpose of performing these studies. In fibroblasts we not only performed genetic studies but also determined enzymatic activities with good correlation between both, which gives more strength to the results. Relatively large deviations are often observed in real-time PCR quantification, owing to the low specificity of the probes and variability of the endogenous controls. However, despite these difficulties, the same expression levels in the first female patient and her brother were observed, which is in agreement with the sequencing results, the low enzymatic activity, the severe clinical presentation and the skewed X-inactivation pattern. The second female showed expression of both mutant and wild-type alleles, which is also in agreement with sequencing results, normal enzymatic activity, slight clinical presentation and random X-inactivation pattern. In conclusion, our results are adequately supported by the studies in controls and are confirmed by the studies in patients. We thank He et al for giving us the opportunity to clarify some issues, although we think that they do not change the conclusions of our study.

17β-Hydroxysteroid dehydrogenase 10 (HSD10) is a mitochondrial enzyme involved in the degradation pathway of isoleucine and branched-chain fatty acids. The gene encoding HSD10, HSD17B10, has been reported as one of the few genes that escapes X-inactivation. We previously studied two female patients with HSD10 deficiency, one of them was severely affected and the other presented a mild phenotype. To elucidate as to why these two carriers were so differently affected, cDNA analyses were performed. The HSD17B10 cDNA of eight control cell lines, two hemizygous patients and two carriers was obtained from cultured fibroblasts, amplified by PCR and sequenced by standard methods. All HSD17B10 cDNAs were quantified by real-time PCR. In the fibroblasts of the female patient who presented with the severe phenotype, only the mutant allele was identified in the cDNA sequence, which was further confirmed by relative quantification (RQ) of HSD17B10 cDNA. This is in agreement with an unfavourable X-inactivation. The other female patient, with slight clinical affectation, showed the presence of both mutant and wild-type alleles in the cDNA sequence, which was confirmed by RQ of HSD17B10 cDNA in fibroblasts. This is in line with normal X-inactivation and the expression of both alleles in different cells (functional mosaicism). RQ results of HSD17B10 cDNA did not differ significantly between male and female controls, which indicate that the genetic doses of mRNA of HSD17B10 was the same in both sexes. In conclusion, these results suggest that the HSD17B10 gene does not escape X-inactivation as has been reported previously.