In vitro dexamethasone treatment does not induce alternative ATM transcripts in cells from Ataxia-Telangiectasia patients.

Short term treatment with low doses of glucocorticoid analogues has been shown to ameliorate neurological symptoms in Ataxia-Telangiectasia (A-T), a rare autosomal recessive multisystem disease that mainly affects the cerebellum, immune system, and lungs. Molecular mechanisms underlying this clinical observation are unclear. We aimed at evaluating the effect of dexamethasone on the induction of alternative ATM transcripts (ATMdexa1). We showed that dexamethasone cannot induce an alternative ATM transcript in control and A-T lymphoblasts and primary fibroblasts, or in an ATM-knockout HeLa cell line. We also demonstrated that some of the reported readouts associated with ATMdexa1 are due to cellular artifacts and the direct induction of γH2AX by dexamethasone via DNA-PK. Finally, we suggest caution in interpreting dexamethasone effects in vitro for the results to be translated into a rational use of the drug in A-T patients.

Mice harbouring a SCA28 patient mutation in AFG3L2 develop late-onset ataxia associated with enhanced mitochondrial proteotoxicity.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Spinocerebellar Ataxia Tethering PCR: A Rapid Genetic Test for the Diagnosis of Spinocerebellar Ataxia Types 1, 2, 3, 6, and 7 by PCR and Capillary Electrophoresis.

Spinocerebellar ataxia (SCA) types 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson disease and amyotrophic lateral sclerosis. Their diagnosis is currently based on a PCR to identify small expanded alleles, followed by a second-level test whenever a false normal homozygous or a CAT interruption in SCA1 needs to be verified. Next-generation sequencing still does not allow efficient detection of these repeats. Here, we show the efficacy of a novel, rapid, and cost-effective method to identify and size pathogenic expansions in SCA1, 2, 3, 6, and 7 and recognize large alleles or interruptions without a second-level test. Twenty-five healthy controls and 33 expansion carriers were analyzed: alleles migrated consistently in different PCRs and capillary runs, and homozygous individuals were always distinguishable from heterozygous carriers of both common and large (>100 repeats) pathogenic CAG expansions. Repeat number could be calculated counting the number of peaks, except for the largest SCA2 and SCA7 alleles. Interruptions in SCA1 were always visible. Overall, our method allows a simpler, cost-effective, and sensibly faster SCA diagnostic protocol compared with the standard technique and to the still unadapted next-generation sequencing.

A case of Feingold type 2 syndrome associated with keratoconus refines keratoconus type 7 locus on chromosome 13q.

We report on a 58-year old woman with microcephaly, mild dysmorphic features, bilateral keratoconus, digital abnormalities, short stature and mild cognitive delay. Except for keratoconus, the phenotype was suggestive for Feingold syndrome type 2 (FGLDS2, MIM 614326), a rare autosomal dominant disorder described in six patients worldwide, due to the haploinsufficiency of MIR17HG, a micro RNA encoding gene. Karyotype showed a de novo deletion on chromosome 13q, further defined by array-Comparative Genomic Hybridization (a-CGH) to a 17.2-Mb region. The deletion included MIR17HG, as expected by the FGLDS2 phenotype, and twelve genes from the keratoconus type 7 locus. Because our patient presented with keratoconus, we propose she further refines disease genes at this locus. Among previously suggested candidates, we exclude DOCK9 and STK24, and propose as best candidates IPO5, DNAJC3, MBNL2 and RAP2A. In conclusion, we report a novel phenotypic association of Feingold syndrome type 2 and keratoconus, a likely contiguous gene syndrome due to a large genomic deletion on 13q spanning MIR17HG and a still to be identified gene for keratoconus.

Exome sequencing in children of women with skewed X-inactivation identifies atypical cases and complex phenotypes.

BACKGROUND: More than 100 X-linked intellectual disability (X-LID) genes have been identified to be involved in 10-15% of intellectual disability (ID). METHOD: To identify novel possible candidates, we selected 18 families with a male proband affected by isolated or syndromic ID. Pedigree and/or clinical presentation suggested an X-LID disorder. After exclusion of known genetic diseases, we identified seven cases whose mother showed a skewed X-inactivation (>80%) that underwent whole exome sequencing (WES, 50X average depth). RESULTS: WES allowed to solve the genetic basis in four cases, two of which (Coffin-Lowry syndrome, RPS6K3 gene; ATRX syndrome, ATRX gene) had been missed by previous clinical/genetics tests. One further ATRX case showed a complex phenotype including pontocerebellar atrophy (PCA), possibly associated to an unidentified PCA gene mutation. In a case with suspected Lujan-Fryns syndrome, a c.649C>T (p.Pro217Ser) MECP2 missense change was identified, likely explaining the neurological impairment, but not the marfanoid features, which were possibly associated to the p.Thr1020Ala variant in fibrillin 1. Finally, a c.707T>G variant (p.Phe236Cys) in the DMD gene was identified in a patient retrospectively recognized to be affected by Becker muscular dystrophy (BMD, OMIM 300376). CONCLUSION: Overall, our data show that WES may give hints to solve complex ID phenotypes with a likely X-linked transmission, and that a significant proportion of these orphan conditions might result from concomitant mutations affecting different clinically associated genes.

Long-term treatment with thiamine as possible medical therapy for Friedreich ataxia.

Thiamine (vitamin B1) is a cofactor of fundamental enzymes of cell energetic metabolism; its deficiency causes disorders affecting both the peripheral and central nervous system. Previous studies reported low thiamine levels in cerebrospinal fluid and pyruvate dehydrogenase dysfunction in Friedreich ataxia (FRDA). We investigated the effect of long-term treatment with thiamine in FRDA, evaluating changes in neurological symptoms, echocardiographic parameters, and plasma FXN mRNA levels. Thirty-four consecutive FRDA patients have been continuously treated with intramuscular thiamine 100 mg twice a week and have been assessed with the Scale for the Assessment and Rating of Ataxia (SARA) at baseline, after 1 month, and then every 3 months during treatment. Thiamine administration ranged from 80 to 930 days and was effective in improving total SARA scores from 26.6 ± 7.7 to 21.5 ± 6.2 (p < 0.02). Moreover, deep tendon reflexes reappeared in 57 % of patients with areflexia at baseline, and swallowing improved in 63 % of dysphagic patients. Clinical improvement was stable in all patients, who did not show worsening even after 2 years of treatment. In a subgroup of 13 patients who performed echocardiogram before and during treatment, interventricular septum thickness reduced significantly (p < 0.02). Frataxin mRNA blood levels were modestly increased in one-half of treated patients. We suppose that a focal thiamine deficiency may contribute to a selective neuronal damage in the areas involved in FRDA. Further studies are mandatory to evaluate thiamine role on FXN regulation, to exclude placebo effect, to verify our clinical results, and to confirm restorative and neuroprotective action of thiamine in FRDA.

Whole exome sequencing is necessary to clarify ID/DD cases with de novo copy number variants of uncertain significance: Two proof-of-concept examples.

Whole exome sequencing (WES) is a powerful tool to identify clinically undefined forms of intellectual disability/developmental delay (ID/DD), especially in consanguineous families. Here we report the genetic definition of two sporadic cases, with syndromic ID/DD for whom array-Comparative Genomic Hybridization (aCGH) identified a de novo copy number variant (CNV) of uncertain significance. The phenotypes included microcephaly with brachycephaly and a distinctive facies in one proband, and hypotonia in the legs and mild ataxia in the other. WES allowed identification of a functionally relevant homozygous variant affecting a known disease gene for rare syndromic ID/DD in each proband, that is, c.1423C>T (p.Arg377*) in the Trafficking Protein Particle Complex 9 (TRAPPC9), and c.154T>C (p.Cys52Arg) in the Very Low Density Lipoprotein Receptor (VLDLR). Four mutations affecting TRAPPC9 have been previously reported, and the present finding further depicts this syndromic form of ID, which includes microcephaly with brachycephaly, corpus callosum hypoplasia, facial dysmorphism, and overweight. VLDLR-associated cerebellar hypoplasia (VLDLR-CH) is characterized by non-progressive congenital ataxia and moderate-to-profound intellectual disability. The c.154T>C (p.Cys52Arg) mutation was associated with a very mild form of ataxia, mild intellectual disability, and cerebellar hypoplasia without cortical gyri simplification. In conclusion, we report two novel cases with rare causes of autosomal recessive ID, which document how interpreting de novo array-CGH variants represents a challenge in consanguineous families; as such, clinical WES should be considered in diagnostic testing. © 2016 Wiley Periodicals, Inc.

A novel 3q29 deletion associated with autism, intellectual disability, psychiatric disorders, and obesity.

Copy number variation (CNV) has been associated with a variety of neuropsychiatric disorders, including intellectual disability/developmental delay (ID/DD), autism spectrum disorder (ASD), and schizophrenia (SCZ). Often, individuals carrying the same pathogenic CNV display high clinical variability. By array-CGH analysis, we identified a novel familial 3q29 deletion (1.36 Mb), centromeric to the 3q29 deletion region, which manifests with variable expressivity. The deletion was identified in a 3-year-old girl diagnosed with ID/DD and autism and segregated in six family members, all affected by severe psychiatric disorders including schizophrenia, major depression, anxiety disorder, and personality disorder. All individuals carrying the deletion were overweight or obese, and anomalies compatible with optic atrophy were observed in three out of four cases examined. Amongst the 10 genes encompassed by the deletion, the haploinsufficiency of Optic Atrophy 1 (OPA1), associated with autosomal dominant optic atrophy, is likely responsible for the ophthalmological anomalies. We hypothesize that the haploinsufficiency of ATPase type 13A4 (ATP13A4) and/or Hairy/Enhancer of Split Drosophila homolog 1 (HES1) contribute to the neuropsychiatric phenotype, while HES1 deletion might underlie the overweight/obesity. In conclusion, we propose a novel contiguous gene syndrome due to a proximal 3q29 deletion variably associated with autism, ID/DD, psychiatric traits and overweight/obesity.

Array-Comparative Genomic Hybridization Analysis in Fetuses with Major Congenital Malformations Reveals that 24% of Cases Have Pathogenic Deletions/Duplications.

Karyotyping and aCGH are routinely used to identify genetic determinants of major congenital malformations (MCMs) in fetal deaths or terminations of pregnancy after prenatal diagnosis. Pathogenic rearrangements are found with a variable rate of 9-39% for aCGH. We collected 33 fetuses, 9 with a single MCM and 24 with MCMs involving 2-4 organ systems. aCGH revealed copy number variants in 14 out of 33 cases (42%). Eight were classified as pathogenic which account for a detection rate of 24% (8/33) considering fetuses with 1 or more MCMs and 33% (8/24) taking into account fetuses with multiple malformations only. Three of the pathogenic variants were known microdeletion syndromes (22q11.21 deletion, central chromosome 22q11.21 deletion, and TAR syndrome) and 5 were large rearrangements, adding up to >11 Mb per subject and comprising strong phenotype-related genes. One of those was a de novo complex rearrangement, and the remaining 4 duplications and 2 deletions were 130-900 kb in size, containing 1-7 genes, and were classified as variants of unknown clinical significance. Our study confirms aCGH as a powerful technique to ascertain the genetic etiology of fetal major congenital malformations.

An atypical form of AOA2 with myoclonus associated with mutations in SETX and AFG3L2.

BACKGROUND: Hereditary ataxias are a heterogeneous group of neurodegenerative disorders, where exome sequencing may become an important diagnostic tool to solve clinically or genetically complex cases. METHODS: We describe an Italian family in which three sisters were affected by ataxia with postural/intentional myoclonus and involuntary movements at onset, which persisted during the disease. Oculomotor apraxia was absent. Clinical and genetic data did not allow us to exclude autosomal dominant or recessive inheritance and suggest a disease gene. RESULTS: Exome sequencing identified a homozygous c.6292C > T (p.Arg2098*) mutation in SETX and a heterozygous c.346G > A (p.Gly116Arg) mutation in AFG3L2 shared by all three affected individuals. A fourth sister (II.7) had subclinical myoclonic jerks at proximal upper limbs and perioral district, confirmed by electrophysiology, and carried the p.Gly116Arg change. Three siblings were healthy. Pathogenicity prediction and a yeast-functional assay suggested p.Gly116Arg impaired m-AAA (ATPases associated with various cellular activities) complex function. CONCLUSIONS: Exome sequencing is a powerful tool in identifying disease genes. We identified an atypical form of Ataxia with Oculoapraxia type 2 (AOA2) with myoclonus at onset associated with the c.6292C > T (p.Arg2098*) homozygous mutation. Because the same genotype was described in six cases from a Tunisian family with a typical AOA2 without myoclonus, we speculate this latter feature is associated with a second mutated gene, namely AFG3L2 (p.Gly116Arg variant). We suggest that variant phenotypes may be due to the combined effect of different mutated genes associated to ataxia or related disorders, that will become more apparent as the costs of exome sequencing progressively will reduce, amplifying its diagnostics use, and meanwhile proposing significant challenges in the interpretation of the data.

Blood metal levels and related antioxidant enzyme activities in patients with ataxia telangiectasia.

Transition metals are cofactors for a wide range of vital enzymes and are directly or indirectly involved in the response against reactive oxygen species (ROS), which can damage cellular components. Their altered homeostasis has been studied in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), but no data are available on rarer conditions. We aimed at studying the role of essential trace elements in ataxia telangiectasia (A-T), a rare form of pediatric autosomal recessive cerebellar ataxia with altered antioxidant response. We found an increased level of copper (Cu, p=0.0002) and a reduced level of zinc (Zn, p=0.0002) in the blood of patients (n. 16) compared to controls, using inductively coupled plasma mass spectrometry (ICP-MS). Other trace elements involved in the oxidative stress response, such as manganese (Mn) and selenium (Se), were unaltered. Cu/Zn-dependent superoxide dismutase (SOD1) was shown to have a 30% reduction in gene expression and 40% reduction in enzyme activity upon analysis of lymphoblastoid cell lines of patients (Student's t-test, p=0.0075). We also found a 30% reduction of Mn-SOD (SOD2; Student's t-test, p=0.02), probably due to a feedback regulatory loop between the two enzymes. The expression of antioxidant enzymes, such as erythrocyte glutathione peroxidase (GPX1), and SOD2 was unaltered, whereas catalase (CAT) was increased in A-T cells, both at the mRNA level and in terms of enzyme activity (~25%). Enhanced CAT expression can be attributed to the high ROS status, which induces CAT transcription. These results suggest that alterations in essential trace elements and their related enzymes may play a role in the pathogenesis of A-T, although we cannot conclude if altered homeostasis is a direct effect of A-T mutated genes (ATM). Altered homeostasis of trace elements may be more prevalent in neurodegenerative diseases than previously thought, and it may represent both a biomarker and a generic therapeutic target for different disorders with the common theme of altered antioxidant enzyme responses associated with an unbalance of metals.

Two families with novel missense mutations in COL4A1: When diagnosis can be missed.

Mutations in COL4A1, encoding one of the six collagen type IV proteins, cover a wide spectrum of autosomal dominant overlapping phenotypes including porencephaly, small-vessel disease and hemorrhagic stroke, leukoencephalopathy, hereditary angiopathy with nephropathy, aneurysms and muscle cramp (HANAC) syndrome, and Walker-Warburg syndrome. Over 50 mutations are known, mainly being missense changes. Intra- and inter-familial variability has been reported. We studied two Italian families in which the proband had a clinical diagnosis of COL4A1-related disorder. We found two novel mutations (c.1249G>C; p.Gly417Arg and c.2662G>C; p.Gly888Arg). Both involved highly conserved amino acids and were predicted as being deleterious by bioinformatics tools. The c.1249G>C (p.Gly417Arg) segregated in four subjects with variable neurological phenotypes, namely leukoencephalopathy with muscle symptoms, brain small-vessel disease, and mild infantile encephalopathy. A fourth case was a carrier of the mutation without any neurological symptoms and an MRI with a specific white matter anomaly. The c.2662G>C (p.Gly888Arg) mutation was de novo in the proband. After a temporary motor impairment at age 14, the subject complained of mild imbalance at age 30, during the third trimester of her twin pregnancy, when an anomaly of the left brain hemisphere was documented in one fetus. Both her male dizygotic twins presented a severe motor delay, early convulsions, and leukoencephalopathy, and were carriers of the mutation. In summary, we confirm that high intra-familial variability of COL4A1 mutations with very mild phenotypes, the apparent incomplete penetrance, and de novo changes may become a "dilemma" for clinicians and genetic counselors.

Adult-onset autosomal recessive ataxia associated with neuronal ceroid lipofuscinosis type 5 gene (CLN5) mutations.

Autosomal recessive inherited ataxias are a growing group of genetic disorders. We report two Italian siblings presenting in their mid-50s with difficulty in walking, dysarthria and progressive cognitive decline. Visual loss, ascribed to glaucoma, manifested a few years before the other symptoms. Brain MRI showed severe cerebellar atrophy, prevalent in the vermis, with marked cortical atrophy of both hemispheres. Exome sequencing identified a novel homozygous mutation (c.935G > A;p.Ser312Asn) in the ceroid neuronal lipofuscinosis type 5 gene (CLN5). Bioinformatics predictions and in vitro studies showed that the mutation was deleterious and likely affects ER-lysosome protein trafficking. Our findings support CLN5 hypomorphic mutations cause autosomal recessive cerebellar ataxia, confirming other reports showing CLN mutations are associated with adult-onset neurodegenerative disorders. We suggest CLN genes should be considered in the molecular analyses of patients presenting with adult-onset autosomal recessive cerebellar ataxia.

Large cryptic genomic rearrangements with apparently normal karyotypes detected by array-CGH.

BACKGROUND: Conventional karyotyping (550 bands resolution) is able to identify chromosomal aberrations >5-10 Mb, which represent a known cause of intellectual disability/developmental delay (ID/DD) and/or multiple congenital anomalies (MCA). Array-Comparative Genomic Hybridization (array-CGH) has increased the diagnostic yield of 15-20%. RESULTS: In a cohort of 700 ID/DD cases with or without MCA, including 15 prenatal diagnoses, we identified a subgroup of seven patients with a normal karyotype and a large complex rearrangement detected by array-CGH (at least 6, and up to 18 Mb). FISH analysis could be performed on six cases and showed that rearrangements were translocation derivatives, indistinguishable from a normal karyotype as they involved a similar band pattern and size. Five were inherited from a parent with a balanced translocation, whereas two were apparently de novo. Genes spanning the rearrangements could be associated with some phenotypic features in three cases (case 3: DOCK8; case 4: GATA3, AKR1C4; case 6: AS/PWS deletion, CHRNA7), and in two, likely disease genes were present (case 5: NR2F2, TP63, IGF1R; case 7: CDON). Three of our cases were prenatal diagnoses with an apparently normal karyotype. CONCLUSIONS: Large complex rearrangements of up to 18 Mb, involving chromosomal regions with similar size and band appearance may be overlooked by conventional karyotyping. Array-CGH allows a precise chromosomal diagnosis and recurrence risk definition, further confirming this analysis as a first tier approach to clarify molecular bases of ID/DD and/or MCA. In prenatal tests, array-CGH is confirmed as an important tool to avoid false negative results due to karyotype intrinsic limit of detection.

SETX mutations are a frequent genetic cause of juvenile and adult onset cerebellar ataxia with neuropathy and elevated serum alpha-fetoprotein.

OBJECTIVES/BACKGROUND: Ataxia with oculomotor apraxia defines a group of genetically distinct recessive ataxias including ataxia-telangectasia (A-T, ATM gene), ataxia with oculomotor apraxia type 1 (AOA1, APTX gene) and type 2 (AOA2, SETX gene). Although, a few unique clinical features differentiate each of these forms, the patients also share common clinical signs, such as the presence of cerebellar atrophy, sensorimotor axonal neuropathy, and elevated alpha-fetoprotein (AFP) serum level. MATERIALS AND METHODS: We selected 22 Italian patients from 21 families, presenting progressive cerebellar ataxia, axonal neuropathy, and elevated serum AFP. We screened the coding regions of ATM, APTX and SETX genes for point mutations by direct sequencing or DHPLC, and searched genomic rearrangements in SETX by MLPA analysis. In selected cases, quantification of ATM and senataxin proteins was performed by Western blot. Clinical, neurophysiological, and neuroimaging data were collected. RESULTS: Thirteen patients (12 families) carried SETX mutations (AOA2, 57%), two were mutated in ATM (A-T), and three in APTX (AOA1). In three remaining patients, we could not find pathogenic mutations, and in one case we found, in homozygosis, the SETX p.K992R polymorphism (population frequency 1-2%). In AOA2 cases, we identified 14 novel and three reported SETX mutations. Signs at onset were gait ataxia and facial dyskinesia, and the age ranged between 11 and 18 years. None had obvious oculomotor apraxia at the latest examination (age 14-45 years). The patient carrying the p.K992R SETX polymorphism had a phenotype similar to that of the diagnosed AOA2 patients, while the other three undiagnosed subjects had a very late onset and a few distinguishing clinical features. DISCUSSION AND CONCLUSIONS: We describe a large series of 13 AOA2 Italian patients. The phenotype was consistent with previous descriptions of AOA2, except for a higher frequency of strabism, and for the absence of oculomotor apraxia. In our survey ~60% of juvenile-to-adult cases with cerebellar ataxia, sensorimotor neuropathy and increased AFP are due to mutations in the SETX gene, and a smaller percentage to APTX and ATM gene mutations.

A de novo X;8 translocation creates a PTK2-THOC2 gene fusion with THOC2 expression knockdown in a patient with psychomotor retardation and congenital cerebellar hypoplasia.

BACKGROUND AND AIM: We identified a balanced de novo translocation involving chromosomes Xq25 and 8q24 in an eight year-old girl with a non-progressive form of congenital ataxia, cognitive impairment and cerebellar hypoplasia. METHODS AND RESULTS: Breakpoint definition showed that the promoter of the Protein Tyrosine Kinase 2 (PTK2, also known as Focal Adhesion Kinase, FAK) gene on chromosome 8q24.3 is translocated 2 kb upstream of the THO complex subunit 2 (THOC2) gene on chromosome Xq25. PTK2 is a well-known non-receptor tyrosine kinase whereas THOC2 encodes a component of the evolutionarily conserved multiprotein THO complex, involved in mRNA export from nucleus. The translocation generated a sterile fusion transcript under the control of the PTK2 promoter, affecting expression of both PTK2 and THOC2 genes. PTK2 is involved in cell adhesion and, in neurons, plays a role in axonal guidance, and neurite growth and attraction. However, PTK2 haploinsufficiency alone is unlikely to be associated with human disease. Therefore, we studied the role of THOC2 in the CNS using three models: 1) THOC2 ortholog knockout in C.elegans which produced functional defects in specific sensory neurons; 2) Thoc2 knockdown in primary rat hippocampal neurons which increased neurite extension; 3) Thoc2 knockdown in neuronal stem cells (LC1) which increased their in vitro growth rate without modifying apoptosis levels. CONCLUSION: We suggest that THOC2 can play specific roles in neuronal cells and, possibly in combination with PTK2 reduction, may affect normal neural network formation, leading to cognitive impairment and cerebellar congenital hypoplasia.

Deep-intronic ATM mutation detected by genomic resequencing and corrected in vitro by antisense morpholino oligonucleotide (AMO).

Recent development of next-generation DNA sequencing (NGS) techniques is changing the approach to search for mutations in human genetic diseases. We applied NGS to study an A-T patient in which one of the two expected mutations was not found after DHPLC, cDNA sequencing and MLPA screening. The 160-kb ATM genomic region was divided into 31 partially overlapping fragments of 4-6 kb and amplified by long-range PCR in the patient and mother, who carried the same mutation by segregation. We identified six intronic variants that were shared by the two genomes and not reported in the dbSNP(132) database. Among these, c.1236-405C>T located in IVS11 was predicted to be pathogenic because it affected splicing. This mutation creates a cryptic novel donor (5') splice site (score 1.00) 405 bp upstream of the exon 12 acceptor (3') splice site. cDNA analysis showed the inclusion of a 212-bp non-coding 'pseudoexon' with a premature stop codon. We validated the functional effect of the splicing mutation using a minigene assay. Using antisense morpholino oligonucleotides, designed to mask the cryptic donor splice-site created by the c.1236-405C>T mutation, we abrogated the aberrant splicing product to a wild-type ATM transcript, and in vitro reverted the functional ATM kinase impairment of the patients' lymphoblasts. Resequencing is an effective strategy for identifying rare splicing mutations in patients for whom other mutation analyses have failed (DHPLC, MLPA, or cDNA sequencing). This is especially important because many of these patients will carry rare splicing variants that are amenable to antisense-based correction.

De novo 13q12.3-q14.11 deletion involving BRCA2 gene in a patient with developmental delay, elevated IgM levels, transient ataxia, and cerebellar hypoplasia, mimicking an A-T like phenotype.

We report on a child with a de novo deletion of approximately 12 Mb detected through array comparative genomic hybridization (CGH). The deletion involved chromosome bands 13q12.3-13q14.11 and determined the loss of ≥50 genes. A second deletion on chromosome 12p11.3p11.22 of 43-167 kb, including about 12 genes, was unlikely of clinical relevance because inherited from the asymptomatic father. The child had developmental delay, dysmorphisms, and many features reminiscent of ataxia-telangiectasia (A-T), as cerebellar ataxia, oculocutaneus telangiectasia, and recurrent upper airway infections. Atraumatic fractures of the metatarsus were noted. Moreover, this is a rare case of 13q deletion syndrome associated with peripheral blood white cells radiosensitivity to bleomycin, reminiscent of what previously reported on X-ray hypersensitivity of fibroblasts from patients with alterations of this chromosome. The immunological evaluation revealed increased IgM serum levels and a low proliferative response to mitogens, PHA, and CD3 cross-linking (CD3 XL). After 12 years of age only a mild dysmetria persisted, while the proliferative response to mitogens became normal by 9 years of age.

Megalencephalic leukoencephalopathy with subcortical cysts type 1 (MLC1) due to a homozygous deep intronic splicing mutation (c.895-226T>G) abrogated in vitro using an antisense morpholino oligonucleotide.

Megalencephalic leukoencephalopathy with subcortical cysts is an autosomal recessive disease characterized by early onset macrocephaly; developmental delay; motor disability in the form of progressive spasticity and ataxia; seizures; cognitive decline; and characteristic magnetic resonance imaging findings. Mutations in two genes, MLC1 (22q13.33; 75 % of patients) or HEPACAM (11q24; 20 % of patients), are associated with the disease. We describe an adult MLC patient with moderate clinical symptoms. MLC1 cDNA analysis from lymphoblasts showed a strong transcript reduction and identified a 246-bp pseudoexon containing a premature stop codon between exons 10 and 11, due to a homozygous c.895-226 T>G deep-intronic mutation. This category of mutations is often overlooked, being outside of canonically sequenced genomic regions. The mutation c.895-226 T>G has a leaky effect on splicing leaving part of the full-length transcript. Its role on splicing was confirmed using a minigene assay and an antisense morpholinated oligonucleotide targeted to the aberrant splice site in vitro, which partially abrogated the mutation effect.