Expansion of the QARS deficiency phenotype with report of a family with isolated supratentorial brain abnormalities.

We describe a family with QARS deficiency due to compound heterozygous QARS mutations, including c.1387G > A (p.R463*) in the catalytic core domain and c.2226C > G (p.Q742H) in the anticodon domain, both previously unreported and predicted damaging. The phenotype of the male index further confirms this specific aminoacyl-transfer RNA (tRNA) synthetase disorder as a novel genetic cause of progressive microcephaly with diffuse cerebral atrophy, severely deficient myelination, intractable seizures, and developmental arrest. However, in contrast to the two hitherto published families, the cerebellum and its myelination are not affected. An awareness that QARS mutations may cause isolated supratentorial changes is crucial for properly directing genetic analysis.

Inferior olivary nucleus involvement in pediatric neurodegenerative disorders: does it play a role in neuroimaging pattern-recognition approach?

The diagnostic work up of neurometabolic/degenerative disorders is complex. In such context, identification of neuroradiological features suggestive of specific diagnoses is useful to prompt further diagnostic tests. Involvement of the inferior olivary nucleus (ION) has been reported in several pathologic conditions, either as a primary manifestation of disease or secondary to hypertrophic olivary degeneration (HOD). In this study, we analyzed a cohort of 95 children with different neurometabolic/degenerative diseases involving the brainstem and cerebellum, with the aim to evaluate whether ION involvement plays a role in a neuroimaging-based pattern-recognition approach. A total of 13 patients (13.7%) showed bilateral high-signal intensity and enlargement of the ION on T2-weighted images, while 16 (16.8%) had ION T2-hyperintensity without olivary nucleus enlargement. Our study demonstrates that ION involvement is not rare in children with neurometabolic/degenerative disorders. Two main neuroradiological patterns, that is, "T2-hyperintense signal" and "T2-hyperintense signal with enlargement" are found. These patterns can be related to different etiologies, and do not suggest specific diagnoses. Primary ION lesion can be characterized by olivary swelling, and the differentiation from typical secondary HOD may be difficult.

A novel homozygous MCOLN1 double mutant allele leading to TRP channel domain ablation underlies Mucolipidosis IV in an Italian Child.

Mucolipidosis type IV (MLIV) is a very rare disorder of late endosome/lysosome transport, characterized by neurodevelopmental abnormalities and progressive visual impairment owing to corneal clouding and retinal dystrophy. Greater than 70 % of MLIV patients are of Ashkenazi Jewish ancestry. Here we report a novel MCOLN1double mutant allele [c.395_397delCTG;c.468_474dupTTGGACC] which introduces a premature stop codon [p.Ala132del; p.Asn159LeufsX27] leading to almost complete abrogation of the region coding mucolipin-1, a member of the transient receptor potential (TRP) cation channel family. The genomic lesion was identified in homozygous state, in a non-Jewish Italian MLIV patient, who also presented abnormal serum gastrin levels. Conventional and advanced MRI sequences, including diffusion tensor imaging and tractography, were used for the assessment of white matter involvement in the patient.

Deficiency of hyccin, a newly identified membrane protein, causes hypomyelination and congenital cataract.

We describe a new autosomal recessive white matter disorder ('hypomyelination and congenital cataract') characterized by hypomyelination of the central and peripheral nervous system, progressive neurological impairment and congenital cataract. We identified mutations in five affected families, resulting in a deficiency of hyccin, a newly identified 521-amino acid membrane protein. Our study highlights the essential role of hyccin in central and peripheral myelination.

A novel homozygous splicing mutation in PSAP gene causes metachromatic leukodystrophy in two Moroccan brothers.

Prosaposin (PSAP) gene mutations, affecting saposin B (Sap-B) domain, cause a rare metachromatic leukodystrophy (MLD) variant in which arylsulfatase A (ARSA) activity is normal. To date, only 10 different PSAP mutations have been associated with a total of 18 unrelated MLD patients worldwide. In this study, we report for the first time a family with Moroccan origins in which the proband, presenting with a late-infantile onset of neurological involvement and a brain MRI with the typical tigroid MLD pattern, showed normal values of ARSA activity in the presence of an abnormal pattern of urinary sulfatides. In view of these findings, PSAP gene was analyzed, identifying the newly genomic homozygous c.909 + 1G > A mutation occurring within the invariant GT dinucleotide of the intron 8 donor splice site. Reverse transcriptase-polymerase chain reaction (RT-PCR), showing the direct junction of exon 7 to exon 9, confirmed the skipping of the entire exon 8 (p.Gln260_Lys303) which normally contains two cysteine residues (Cys271 and Cys265) involved in disulfide bridges. Our report provides further evidence that phenotypes of patients with Sap-B deficiency vary widely depending on age of onset, type, and severity of symptoms. Awareness of this rare MLD variant is crucial to prevent delayed diagnosis or misdiagnosis and to promptly provide an accurate genetic counseling, including prenatal diagnosis, to families.

Further genotype-phenotype correlation emerging from two families with PLP1 exon 4 skipping.

Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus-Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and 'PLP1 null syndrome'), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype-phenotype correlations of patients with PLP1 splice-site mutations.

Periventricular nodular heterotopia in Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is caused by an interstitial microdeletion of chromosome 17p11.2. A few patients with the typical SMS phenotype have RAI1 gene mutations. The syndrome is characterized by minor craniofacial anomalies, short stature, sleep disturbances, behavioural and neurocognitive abnormalities, as well as variable multisystemic manifestations. Periventricular nodular heterotopia (PNH) is a genetically heterogeneous neuronal migration disorder characterized by subependymal heterotopic nodules, and is variably associated with other brain malformations, epileptic seizures and intellectual disability. Here we report on two patients harboring deletions of the 17p11.2 region in whom the SMS typical phenotype was associated with bilateral PNH. Our observations expand the spectrum of chromosomal rearrangements associated with PNH and indicate that abnormal neuronal migration may contribute to the neurocognitive phenotype of SMS.

Magnetic resonance imaging "tigroid pattern" in Alexander disease.

Alexander disease (AD) is a rare white matter disorder resulting from mutations in the gene encoding for the glial fibrillary acidic protein. Diffuse white matter involvement with frontal predominance is typical of infantile AD that is clinically characterized by progressive motor and mental retardation, seizures, and megaloencephaly. We describe the case of a 10-year-old patient harboring a de novo missense mutation c.235C > T (p.R79C) in the GFAP gene, showing a relatively slow clinical and neuroradiologic progression of disease associated with a previously unreported magnetic resonance imaging (MRI) finding consistent with the so-called tigroid pattern. This pattern has been previously described in only a few different neurologic conditions, including Pelizaeus-Merzbacher disease and some lysosomal disorders. This report expands the spectrum of MRI features in AD.

TRPV4 mutations in children with congenital distal spinal muscular atrophy.

Inherited disorders characterized by motor neuron loss and muscle weakness are genetically heterogeneous. The recent identification of mutations in the gene encoding transient receptor potential vanilloid 4 (TRPV4) in distal spinal muscular atrophy (dSMA) prompted us to screen for TRPV4 mutations in a small group of children with compatible phenotype. In a girl with dSMA and vocal cord paralysis, we detected a new variant (p.P97R) localized in the cytosolic N-terminus of the TRPV4 protein, upstream of the ankyrin-repeat domain, where the great majority of disease-associated mutations reside. In another child with congenital dSMA, in this case associated with bone abnormalities, we detected a previously reported mutation (p.R232C). Functional analysis of the novel p.P97R mutation in a heterologous system demonstrated a loss-of-function mechanism. Protein localization studies in muscle, skin, and cultured skin fibroblasts from both patients showed normal protein expression. No TRPV4 mutations were detected in four children with dSMA without bone or vocal cord involvement. Adding to the clinical and molecular heterogeneity of TRPV4-associated diseases, our results suggest that molecular testing of the TRPV4 gene is warranted in cases of congenital dSMA with bone abnormalities and vocal cord paralysis.

Intermittent-relapsing pyruvate dehydrogenase complex deficiency: a case with clinical, biochemical, and neuroradiological reversibility.

Pyruvate dehydrogenase complex (PDHC) deficiency causes encephalomyopathies, of which there are four major categories: (1) neonatal encephalopathy with lactic acidosis; (2) an early infantile form, which (3) at times resembles Leigh syndrome; and (4) a later-onset form. Long-term clinical and radiological follow-up is still incompletely elucidated. We report a 12-year-old male with intermittent-relapsing PDHC deficiency who presented with three typical acute episodes of metabolic decompensation over 7 years. Neuroimaging showed reversible signal abnormalities in the basal ganglia, inferior olivary nuclei, periaqueductal grey matter, and dentate nuclei, with evidence of lactate on magnetic resonance spectroscopy. Molecular analysis of PDH1A revealed a novel hemizygous c.1045G>A mutation, predicting a p.A349T missense mutation. He was treated with thiamine supplementation and, while on this regimen, he experienced several intercurrent febrile episodes without neurological compromise. This case report stresses the importance of performing neuroimaging during acute clinical episodes because brain lesions in PDHC deficiency may be transient and reversible, and false-negative results may mislead the diagnosis and delay the treatment.

Cerebellar hypoplasia and brainstem thinning associated with severe white matter and basal ganglia abnormalities in a child with an mtDNA deletion.

Cerebellar and brainstem hypoplasia may occur in different conditions, including those disorders designated as pontocerebellar hypoplasia (PCH). In particular, when PCH is combined with severe supratentorial white matter involvement and cerebral atrophy, mutations in the mitochondrial arginyl-tRNA synthethase (RARS2) gene causing PCH6 are possible. We describe a patient with a lethal mitochondrial encephalomyopathy due to a mtDNA deletion and no alterations in RARS2, whose magnetic resonance (MR) findings mimicked PCH6. A thorough diagnostic work-up for mitochondrial disorders should be carried out when facing with a PCH-like and severe white matter and basal ganglia involvement on brain MR imaging in children, even if clinical and laboratory mitochondrial "stigmata" are scant or nonspecific.

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease.

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.

PLP1 gene duplication causes overexpression and alteration of the PLP/DM20 splicing balance in fibroblasts from Pelizaeus-Merzbacher disease patients.

The PLP1 gene encodes two protein isoforms (PLP and DM20) which represent the predominant protein portion in myelin of the central nervous system. The two products are generated from the same primary transcript by alternative splicing. Defects of the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) or X-linked spastic paraplegia type 2 (SPG2). Duplication of the PLP1 gene is the most frequent gene defect, usually responsible for the classic form of PMD. To investigate the effects of PLP1 gene over dosage on gene expression, we analysed the PLP/DM20 expression profile in fibroblasts from three PMD patients with a PLP1 gene duplication. Gene expression was evaluated by real-time PCR using two different PLP1 amplicons and two different reference genes (GAPDH and GUSB). Fibroblasts from the three patients showed a 4-5 fold increase of PLP1 gene expression compared to fibroblasts from three normal controls. The contribution of the two alternatively spliced transcript isoforms (PLP and DM20) to the whole PLP1 gene expression was investigated using a DM20-specific amplicon. The three patients showed a decrease of the DM20/(DM20+PLP) ratio in comparison to the three normal controls, suggesting a prominent contribution of the PLP transcript to the PLP1 gene overexpression detected in the patients. Therefore, PLP1 gene duplication seems to result both in overexpression and in a shift of the PLP/DM20 splicing balance in direction of the PLP isoform.

A novel polymorphic AP-1 binding element of the GFAP promoter is associated with different allelic transcriptional activities.

The Glial Fibrillary Acidic Protein (GFAP) gene encodes a cytoskeletal protein belonging to the intermediate filament family whose expression is considered as a marker of astrocytes differentiation. GFAP expression, shown to be upregulated as a consequence of brain gliosis, depends on hormones, growth factors, cytokine, and transcription factors and, among these latters, activator protein 1 (AP-1) has been demonstrated to play a crucial role. In this study, we have focused on a 2.2 kb sequence of the regulatory region located upstream of the GFAP gene, searching in a panel of control individuals for single-nucleotide polymorphisms (SNPs) that could modulate GFAP transcription. Among four SNPs of the GFAP promoter whose alleles have been predicted by in silico analysis to induce differences in the pattern of binding transcription factors, we have identified a new AP-1 binding site lying at -250 bp upstream from the GFAP transcriptional start site. The two alleles of this polymorphic locus have shown to bind the AP-1 complex to different extents, thus promoting variable transcriptional activities of the GFAP promoter. Therefore, these SNP alleles may, among others, mediate the effects of GFAP mutations, thus explaining the phenotypic heterogeneity of Alexander disease.

Anti-N-methyl-D-aspartate-receptor encephalitis in a four-year-old girl.

Anti-N-methyl-D-aspartate-receptor encephalitis is a recently identified autoimmune disorder. We report on a 4-year-old girl presenting with seizures after nonspecific viral-like symptoms, progressing to severe aphasia, upper limb dyskinesias, fluctuation in consciousness, and inability to walk. Anti-N-methyl-D-aspartate-receptor encephalitis should be included in the differential diagnosis of acute/subacute encephalitis in children.

Microcephaly, sensorineural deafness and Currarino triad with duplication-deletion of distal 7q.

Currarino syndrome (CS) is a peculiar form of caudal regression syndrome [also known as autosomal dominant sacral agenesis (OMIM no. 176450)] characterised by (1) partial absence of the sacrum with intact first sacral vertebra, (2) a pre-sacral mass and (3) anorectal anomalies (Currarino triad). We studied a 3-year-old girl with Currarino triad who had additional systemic features and performed array comparative genomic hybridisation to look for chromosomal abnormalities. This girl had the typical spectrum of anomalies of the CS including (a) partial sacral agenesis (hemisacrum with remnants of only sacral S1-S2 vertebrae and a residual S3 vertebral body) associated with complete coccygeal agenesis, (b) pre-intrasacral dermoid, (c) intra-dural lipoma, (d) ectopic anus and (e) tethered cord. She had, in addition, pre- and post-natal growth impairment (<3rd percentile), severe microcephaly (<-3 SD) with normal gyration pattern and lack of cortical thickening associated with a hypoplastic inferior vermis, facial dysmorphism, sensorineural deafness and decreased serum levels of IGF-1. A de novo 10.3-Mb duplication of 7q34-q35 and an 8.8-Mb deletion on 7q36 were identified in this patient. The Homeobox HLXB9 (CS) gene is contained within the deletion accounting for the CS phenotype including microcephaly. The spectrums of associated abnormalities in the IGF-1 deficiency growth retardation with sensorineural deafness and mental retardation syndrome (OMIM no. 608747) are discussed. To the best of our knowledge, this is the first reported case of a patient with distal 7q chromosomal imbalance and features of CS triad (including microcephaly) and the first documented case of a patient with normal gyration pattern microcephaly. The spectrum of associated anomalies in this newly recognised phenotype complex consists of growth failure, typical facial anomalies with additional (previously unreported) nervous system abnormalities (e.g. sensorineural deafness) and somatomedin C deficiency.

Mutations in MFSD8/CLN7 are a frequent cause of variant-late infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCL) are a group of genetically heterogeneous neurodegenerative disorders. The recent identification of the MFSD8/CLN7 gene in a variant-late infantile form of NCL (v-LINCL) in affected children from Turkey prompted us to examine the relative frequency of variants in this gene in Italian patients with v-LINCL. We identified nine children harboring 11 different mutations in MFSD8/CLN7. Ten mutations were novel and included three nonsense (p.Arg35Stop, p.Glu381Stop, p.Arg482Stop), four missense (p.Met1Thr, p.Gly52Arg, p.Thr294Lys, p.Pro447Leu), two splice site mutations (c.863+3_4insT, c.863+1G>C), and a 17-bp deletion predicting a frameshift and premature protein truncation (c.627_643del17/p.Met209IlefsX3). The clinical phenotype, which was similar to that of the Turkish v-LINCL cases, was not influenced by type and location of the mutation nor the length of the predicted residual gene product. As well as identifying novel variants in MFSD8/CLN7, this study contributes to a better molecular characterization of Italian NCL cases, and will facilitate medical genetic counseling in such families. The existence of a subset of v-LINCL cases without mutations in any of the known NCL genes suggests further genetic heterogeneity.

Screening of ARHSP-TCC patients expands the spectrum of SPG11 mutations and includes a large scale gene deletion.

Autosomal recessive spastic paraplegia with thinning of corpus callosum (ARHSP-TCC) is a complex form of HSP initially described in Japan but subsequently reported to have a worldwide distribution with a particular high frequency in multiple families from the Mediterranean basin. We recently showed that ARHSP-TCC is commonly associated with mutations in SPG11/KIAA1840 on chromosome 15q. We have now screened a collection of new patients mainly originating from Italy and Brazil, in order to further ascertain the spectrum of mutations in SPG11, enlarge the ethnic origin of SPG11 patients, determine the relative frequency at the level of single Countries (i.e., Italy), and establish whether there is one or more common mutation. In 25 index cases we identified 32 mutations; 22 are novel, including 9 nonsense, 3 small deletions, 4 insertions, 1 in/del, 1 small duplication, 1 missense, 2 splice-site, and for the first time a large genomic rearrangement. This brings the total number of SPG11 mutated patients in the SPATAX collection to 111 cases in 44 families and in 17 isolated cases, from 16 Countries, all assessed using homogeneous clinical criteria. While expanding the spectrum of mutations in SPG11, this larger series also corroborated the notion that even within apparently homogeneous population a molecular diagnosis cannot be achieved without full gene sequencing.

Variant late infantile ceroid lipofuscinoses associated with novel mutations in CLN6.

The neuronal ceroid lipofuscinoses (NCL) are heterogeneous neurodegenerative disorders with typical autofluorescence material stored in tissues. Ten clinical NCL forms and eight causative genes are known. Mutations in CLN6 have been reported in roughly 30 patients, mostly in association with the variant late-infantile NCL (v-LINCL) phenotype. We screened CLN6 in 30 children from a cohort of 53 v-LINCL cases and revised their clinical and ultrastructural features. We detected 11 mutations, eight of which are novel, all predicting a direct impairing of the putative gene function. No clear-cut genotype-phenotype correlations were observed, with inter- and intra-familial variability evident for few recurrent mutations. Ultrastructural findings were suggestive of an impaired regulation of the autophagic vacuoles turnover. While expanding the array of CLN6 mutations, we showed that more than half of our v-LINCL cases lack a DNA confirmation and further molecular etiologies are to be searched.