Simona Cavalieri

A rapid flow cytometry test based on histone H2AX phosphorylation for the sensitive and specific diagnosis of ataxia telangiectasia

Ataxia telangiectasia (A‐T) is a progressive neurodegenerative disease with onset in early childhood, caused by mutations in the ATM (ataxia‐telangiectasia mutated) gene. Diagnosis relies on laboratory tests showing high levels of serum alphafetoprotein, cell sensitivity to ionizing radiation (IR) and absence or reduced levels of ATM protein. Many tests, however, are not sufficiently sensitive or specific for A‐T, have long turnaround times, or require large blood samples. This prompted us to develop a new flow cytometry method for the diagnosis of A‐T based on the measurement of histone H2AX phosphorylation. We established normal ranges of histone H2AX phosphorylation after 2 Gy IR by testing T‐cell lines, lymphoblastoid cell lines (LCLs) and/or peripheral blood mononuclear cells (PBMCs) or both from 20 genetically proven A‐T and 46 control donors. To further evaluate the specificity and sensitivity of the test, we analyzed cells from 19 patients suspected of having A‐T, and from one Friedreich Ataxia, one Ataxia with Oculomotor Apraxia type 2, and one Nijmegen Breakage Syndrome patients. Phosphorylated histone H2AX mean fluorescence intensity of irradiated A‐T cells was significantly lower than that of healthy donors. The intrastaining, intraassay, and interassay imprecisions were ≤13.22%. Sensitivity and specificity were virtually 100% when the test was performed on PBMCs. Screening of 19 consecutive new patients with suspected A‐T classified 15 patients as non‐A‐T and four as A‐T; diagnosis of the latter four was subsequently confirmed by DNA sequencing to identify ATM mutations. The Friedreich Ataxia patient, the Ataxia with Oculomotor Apraxia type 2 patient and the Nijmegen Breakage Syndrome patient were classified as non‐A‐T. This flow cytometry test is very sensitive, specific and rapid, and requires only 2 ml of blood. It may thus be proposed for the early differential diagnosis of A‐T as an alternative to methods requiring the production of LCLs.

A family with autosomal dominant leukodystrophy linked to 5q23.2–q23.3 without lamin B1 mutations

Background and purpose:  Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult‐onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD.

Functional Characterization and Targeted Correction of ATM Mutations Identified in Japanese Patients with Ataxia- Telangiectasia

A recent challenge for investigators studying the progressive neurological disease ataxia‐telangiectasia (A‐T) is to identify mutations whose effects might be alleviated by mutation‐targeted therapies. We studied ATM mutations in eight families of Japanese A‐T patients (JPAT) and were able to identify all 16 mutations. The probands were compound heterozygotes in seven families, and one (JPAT2) was homozygous for a frameshift mutation. All mutations—four frameshift, two nonsense, four large genomic deletions, and six affecting splicing—were novel except for c.748C>T found in family JPAT6 and c.2639‐384A>G found in family JPAT11/12. Using an established lymphoblastoid cell line (LCL) of patient JPAT11, ATM protein was restored to levels approaching wild type by exposure to an antisense morpholino oligonucleotide designed to correct a pseudoexon splicing mutation. In addition, in an LCL from patient JPAT8/9, a heterozygous carrier of a nonsense mutation, ATM levels could also be partially restored by exposure to readthrough compounds (RTCs): an aminoglycoside, G418, and a novel small molecule identified in our laboratory, RTC13. Taken together, our results suggest that screening and functional characterization of the various sorts of mutations affecting the ATM gene can lead to better identification of A‐T patients who are most likely to benefit from rapidly developing mutation‐targeted therapeutic technologies. Hum Mutat 33:198–208, 2012.

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.

Large Genomic Mutations within the ATM Gene Detected by MLPA, Including a Duplication of 41 kb from Exon 4 to 20

Mutation detection remains problematic for large genes, primarily because PCR‐based methodology fails to detect heterozygous deletions and any duplication. In the ATM gene only a handful of multi‐exon deletions have been described to date, and this type of mutation has been considered rare. To address this issue we tested a new MLPA (Multiplex Ligation Probe Amplification) kit that covers 33 of the 66 ATM exons, using for controls two previously characterized genomic deletions in addition to three A‐T patients, taken from a survey of nine, who had missing four mutations unidentified after conventional mutation screening. We identified for the first time: 1) a ∼41 kb genomic duplication spanning exons 4–20 (c.‐30_2816dup41kb)(a.k.a., ATM dup 41 kb); 2) a novel genomic deletion including exon 31, and 3) in hemizygosis a point mutation in the non‐deleted exon 31. In this study we extended mutation detection to nine new Italian A‐T patients, using a combined approach of haplotype analysis, DHPLC and MLPA. Overall we achieved a mutation detection rate of >97%, and can now define a spectrum of ATM mutations based on twenty‐one consecutive Italian families with A‐T.

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

Objectives/backgroundAtaxia 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 methodsWe 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.ResultsThirteen 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 conclusionsWe 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.

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

BackgroundConventional 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%.ResultsIn 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.ConclusionsLarge 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.

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.

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 Alzheimers disease (AD), Parkinsons 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 (Students t-test, p=0.0075). We also found a 30% reduction of Mn-SOD (SOD2; Students 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.