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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the disclosure relates to methods of diagnosing and treating intellectual disability (ID) syndrome, autism and autism related disorders.
• the disclosure provides diagnostic methods based on identifying alterations in the AUTS2 gene, in particular in exons 9- 19 of the AUTS2 gene and preferably in the AUTS2 short isoform.
• the disclosure further relates to therapeutic interventions utilizing the newly identified alternative transcript.
• Autism is a neuropsychiatric disorder characterized by abnormal social behavior and communication skills associated with repetitive behavior. It belongs to the group of pervasive developmental disorders ("autism spectrum” disorders) which includes autism, Asperger syndrome, Childhood disintegrative disorder, and Pervasive
• DSM IV Diagnostic and Statistical Manual of Mental Disorders uses the following criteria to diagnose autism: (I) A total of six (or more) items from (A), (B), and (C), with at least two from (A), and one each from (B) and (C)
• a method is provided of classifying an individual comprising detecting an alteration in an AUTS2 gene of said individual.
• the presence of an alteration in the AUTS2 gene indicates that the individual has or is at risk of developing AUTS2 syndrome.
• the alteration is a chromosome abnormality, more preferably an intragenic deletion.
• said individual is first selected as having at least one symptom of the AUTS2 syndrome.
• AUTS2 syndrome includes autism and intellectual disability.
• a preferred form of intellectual disability (syndrome) is Rett syndrome.
• the alteration is located between exons 9- 19 (inclusive of exons 9 and 19) of the AUTS2 gene.
• the alteration affects the AUTS2 short isoform, e.g., by altering expression or modifying the amino acid sequence.
• the alteration located between exons 9-19 or affecting the AUTS2 short isoform indicates the severity of the AUTS2 syndrome.
• the detection is performed using multiplex ligation dependent probe amplification (MLPA).
• MLPA multiplex ligation dependent probe amplification
• said MLPA is performed using one or more probe pairs selected from MLPA 102, MLPA 110, MLPA 118, MLPA 126, MLPA 130, MLPA 096, MLPA 096, MLPA 136, MLPA 140, MLPA144, MLPA 100, MLPA 108, MLPA 112, MLPA 116, MLPA 120, MLPA 124, MLPA 128, MLPA 132, MLPA 134, MLPA 105, MLPA 138, MLPA 107, and MLPA 136.
• the present disclosure also provides multiplex ligation dependent probe amplification (MLPA) probe sets specific for the AUTS2 gene.
• a polypeptide comprising an AUTS2 sequence having at least 95% identity to SEQ ID NO:2 is provided.
• the N- terminus of said amino acid sequence is encoded by exon 9 of the AUTS2 gene.
• an isolated nucleic acid encoding said polypeptide is provided.
• a vector comprising said nucleic acid is provided provided.
• the vector is for use in gene therapy, preferably in the treatment of AUTS2 syndrome.
• the disclosure further provides a method of treating an individual in need thereof comprising administering a therapeutically effective amount of a composition comprising the polypeptides, nucleic acids, vectors, or cells as described herein.
• an anti-AUTS2 antibody which specifically binds to the AUTS2 short isoform is provided.
• a mammalian cell comprising a nucleic acid or vector as described herein is provided.
• Figure 1 Overview oiAUTS2 aberrations. Overview of all AUTS2 aberrations in the probands that were included in this study. The location of the deletions is indicated by the bars, the inversion breakpoint is indicated by an arrowhead, the area in which the translocation breakpoint is located is indicated by '-' . On the right you can appreciate a visualization of the AUTS2 syndrome severity score, where the darker shades indicate a more severe and/or more specific phenotype (lightest gray: ⁇ 6; light gray: 6- 10; gray: 11-15; dark gray: 16-20; darkest gray >20).
• Figure 2 Pictures of the AUTS2 aberration patients, a: Patient 5 at the age of 3 years shows no evident dysmorphic features, b+i: Patient 8 at the age of 2,5 years has a repaired cleft lip, mild proptosis, short and mild upslanting palpebral fissures, c: The mother of patient 8 also shows a repaired cleft lip, ptosis and retrognatia. d:
• Patient 9 at the age of 3 years shows highly arched eyebrows, mild downslanting palpebral fissures, epicanthal folds and a short philtrum.
• e+m patient 10 at the age of about 6 years. She is hyperteloric, has a ptosis and down slanting palpebral features, a short philtrum and narrow mouth as does her brother shown in f+n at the ages of 10 years
• g+o Patient 13 at the age of 32. You can appreciate hypertelorism, proptosis, upslanting palpebral fissures, a short upturned philtrum an a narrow mouth, h:
• i+p Patient 17 at the age of 5,5 years. He has a hypertelorism, ptosis, a broad nasal bridge, a short and upturned philtrum and a narrow mouth
• j+k+q Patient 19 at the age of about 1 year (j) and 4,8 years (k). He has a broad nasal bridge, short palpebral fissures and a short philtrum and narrow mouth.
• Figure 3 Evolutionary conservation of exons that constitute the main AUTS2 transcript in humans and a novel transcriptional start site (TSS) detected in exon 9 of human AUTS2 using 5'RACE to amplify capped 5' mRNA ends in a human brain mRNA sample.
• TSS transcriptional start site
• AUTS2 orthologs were detected in gorilla (gorGor3), macaque (Mmul_l), dog (Broadd2), cow (Btau_4.0), pig (Sscrofa9), mouse (NCBIM37), chicken (Washuc2), clawed frog (JGI_4.2) and zebrafish (Zf9) by tblastn searches using the human amino acid sequence as a query against the corresponding genome databases, a) Compared exon-intron organization oiAUTS2 orthologs in humans and zebrafish. Red arrows indicate the location of two transcriptional start sites (TSS) used in human brain mRNA. The alternative TSS is located 1, 17 Mbp downstream in the cluster that contains exons 7- 19.
• TSS transcriptional start sites
• Amino acid alignment (determined by ClustalW2) of human AUTS2 orthologs in gorilla, macaque, dog, cow, pig, mouse, chicken, clawed frog and zebrafish. Exon boundaries are indicated above the alignment. Fully conserved or similar residues are marked by symbols at below the alignment. Grey lowercase letters indicate low-complexity or repetitive segments of the human amino acid sequence according to the XNU + SEG algorithms 11 13 .
• FIG. 6 NM_015570 Homo sapiens autism susceptibility candidate 2 (AUTS2) mRNA
• the autism susceptibility candidate 2 (AUTS2) gene was identified in a translocation breakpoint analysis in twins with autism, developmental delay and epilepsy 1 .
• the authors of this study concluded that AUTS2 is unlikely an autism susceptibility gene for idiopathic autism.
• five patients with a translocation breakpoint and one patient with an inversion breakpoint disrupting AUTS2 were described; these patients have intellectual disability (ID) and/or developmental delay (all), autism features (in four reported cases), epilepsy and skeletal abnormalities (reported in three cases each) 2 4 .
• ID intellectual disability
• all autism features
• epilepsy epilepsy
• skeletal abnormalities reported in three cases each
• Disruptions in either neighboring genes or distantly located genes may also be responsible for the patient phenotypes.
• an intragenic deletion in AUTS2 has any affect on neurodevelopment.
• the function of the protein encoded by AUTS2 is largely unknown.
• the longest transcript oiAUTS2 has 19 exons and spans 1.2 Mb.
• the wild-type human AUTS2 gene sequence corresponds to Gene ID: 26053 located on human chromosome 7ql l.22. It encodes a 1,295 amino acid protein that contains 2 potential proline-rich protein- binding domains 1 .
• the mouse Auts2 ortholog is highly expressed in the developing mouse brain, specifically in the frontal cortex, the hippocampus and the cerebellum— areas known to be affected in autistic patients.
• the Auts2 protein is located in the nucleus and co-localizes in the cortex with Tbrl, a transcription factor critical for corticogenesis 8 .
• AUTS2 syndrome The 5' end (exon 1-4) oiAUTS2 was flagged for having the strongest statistical signal for a putative positive selective sweep in early modern humans 7 .
• the frequency of derived alleles for human polymorphic sites is less than expected in Neanderthals.
• the present invention is based, in part, on results which conclusively demonstrate that that disruption of the AUTS2 gene is associated with a complex phenotype that includes not only autism, but also a series of anatomical and neurodevelopmental defects including intellectual disability, which are referred to herein as AUTS2 syndrome.
• Table 1 provides the phenotypes associated with AUTS2 syndrome and their prevalence.
• Embodiments of the invention are also based on the discovery that the AUTS2 gene encodes an alternatively spliced isoform in the human brain. The inventors further demonstrate that disruptions in the genomic region encoding the alternatively spliced isoform are associated with more severe form of AUTS2 syndrome.
• AUTS2 syndrome is characterized by intellectual disability, microcephaly, mild short stature, feeding difficulties, hypotonia, cerebral palsy and dysmorphic features. It is understood that an affected individual will usually not present with all of the symptoms and may show varying degrees of severity for each symptom.
• AUTS2 syndrome is characterized by at least two symptoms selected from intellectual disability, microcephaly, mild short stature, feeding difficulties, hypotonia, cerebral palsy and dysmorphic features.
• AUTS2 syndrome is characterized by at least the presence of autism as defined by the DSM IV.
• AUTS2 syndrome is synonymous with autism.
• the AUTS2 syndrome is synonymous with intellectual disability.
• One aspect of the disclosure provides a method of classifying an individual comprising detecting an alteration in an AUTS2 gene of said individual. Alterations may be detected at the level of DNA, RNA, or protein, preferably said alteration is detected in a nucleic acid. Said methods may detect a change in expression level (including haploinsufficiency caused either by a deletion or by nonsense mediated decay) or change in amino acid sequence.
• Alterations include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence.
• sequence changes can alter the polypeptide encoded by the nucleic acid.
• the change in the nucleic acid sequence causes a frame shift
• the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
• An alteration can also alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of an encoded polypeptide. It can also alter DNA to increase the possibility that structural changes, such as amplifications or deletions, occur at the somatic level. These alterations can be detected by any number of methods known to a skilled person at the level of DNA, RNA, or protein.
• Detection of the expression levels can be performed using immunological assays, including, Western Blot, immunohistochemistry, FACS analysis, radio immuno assay (RIA), immunofluorescence.
• Methods for the detection of particular mRNAs are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for the AUTS2 short isoform, and other amplification type detection methods).
• Chromosome abnormality refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome.
• the term "normal” refers to the predominate structure or nucleic acid sequence found in healthy individuals. Chromosome abnormalities include insertions, deletions, duplications, translocations, and inversions. Preferably, the abnormality is an intragenic deletion of the AUTS2 gene.
• an intragenic deletion is a less severe chromosomal abnormality than, e.g., large deletions or inversions. While duplications, translocations, and inversions, can affect several genes, the effects of an intragenic deletion are normally confined to the specific gene.
• the intragenic deletion is less than 150kb, more preferably less than lOOkb.
• the method of the invention permits the classification of an individual as to the risk of developing AUTS2 syndrome or of the likelihood of suffering from AUTS2 syndrome.
• the method of the invention also permits the classification of an individual as to the risk that an offspring of said individual may be of risk of developing AUTS2 syndrome.
• the current diagnosis of autism and autism related disorders is based on behaviour. This manner of diagnosis suffers from the disadvantage that the symptoms may widely vary among individuals and that the diagnosis can be complicated by other conditions, such as blindness, which can result in difficulties in socializing or communicating.
• the method of the invention provides objective criteria by which to assess the presence or risk of developing AUTS2 syndrome.
• the presence of at least one alteration in the AUTS2 gene is an indicator that the individual is at risk of developing AUTS2 syndrome, is likely suffering from AUTS2 syndrome, or is at risk of producing an offspring having AUTS2 syndrome.
• the assessment of risk of developing or likelihood of suffering from AUTS2 syndrome may also include determining whether the individual has intellectual disability, multiple congenital anomaly (MCA), or difficulties in social skills, language and behaviour that are known to be associated with autism.
• MCA multiple congenital anomaly
• the method of the invention can be used in combination with symptomatic diagnostic criteria, e.g. based on DSM IV autism diagnosis.
• symptomatic diagnostic criteria e.g. based on DSM IV autism diagnosis.
• Both the risk of and likelihood of having AUTS2 syndrome also increase when an individual also exhibits one or more of the following features: microcephaly, mild short stature, feeding difficulties, hypotonia, cerebral palsy and dysmorphic features.
• the risk of and likelihood of having AUTS2 syndrome may also increase when an individual does not have any symptoms, but has a family history of autism.
• any individual may be classified using a method of the invention. It is preferred that said individual is tested as soon as possible, preferably before or at birth. In another preferred embodiment the individual is first selected based on behavioural criteria, such as having at least one symptom of the AUTS2 syndrome.
• behavioural criteria such as having at least one symptom of the AUTS2 syndrome.
• an individual is selected who presents with intellectual disability, multiple congenital anomaly (MCA), or difficulties in social skills, language and behaviour that are known to be associated with autism.
• MCA congenital anomaly
• the individual has been diagnosed with autism using symptomatic diagnostic criteria, e.g. based on DSM IV.
• said individual also exhibits one or more of the following features: microcephaly, mild short stature, feeding difficulties, hypotonia, cerebral palsy and dysmorphic features.
• the AUTS2 syndrome is characterized by intellectual disability and at least one other symptom selected from microcephaly, mild short stature, feeding difficulties, hypotonia, cerebral palsy, and dysmorphic
• the individual does not have any symptoms but has a family history of autism.
• said individuals are humans and may be adults, children, or fetuses.
• a UTS2 encodes, in addition to the full- length message, a shorter isoform in the human brain.
• This shorter isoform uses an alternative transcription start site in the center of exon 9 followed by a start codon in the same reading frame as the full length AUTS2 transcript.
• the sequence of the short isoform is highly conserved across species. Consistent with a critical functional role for this isoform, deletion of this region is associated with significantly more severe phenotypes.
• the nucleic acid coding sequence of the shorter isoform is:
• the protein sequence of the shorter isoform is predicted to be:
• R (SEQ ID NO:2).
• the present disclosure provides methods as described herein for classifying an individual comprising detecting an alteration in the AUTS2 short isoform.
• the AUTS2 short isoform refers to SEQ ID NO:2.
• An alteration includes a mutation affecting the expression and/or amino acid sequence of the AUTS2 short isoform. Alterations indicate an increased risk of developing or having AUTS2 syndrome.
• the methods disclosed herein comprise detecting at least one alteration, preferably a chromosomal abnormality, between exons 9- 19 (inclusive of exons 9 and 19) of the AUTS2 gene of said individual. Exons 9-19 correspond to nucleotide positions 2212-6426 of NM_015570.
• the alteration may affect exons 9-19 or their corresponding introns. These exons encode the AUTS2 short isoform and alterations in either the intervening introns or 5' and 3' genomic regions may affect the expression and/or proper splicing of the short isoform.
• the alteration is an intragenic deletion deleting at least one or a part of exons 9-19.
• the alteration between exons 9-19 of the AUTS2 gene is preferably in the genomic region which encodes the short isoform and/or affects translation or splicing. Identification of an alteration between exons 9-19 or in the genomic region encoding the AUTS2 short isoform may also indicate that the severity of AUTS2 syndrome is likely to be greater than the severity resulting from an abnormality in the 5' region of AUTS2. This finding is contrary to what one would have predicted. A disruption in the 3' end of a gene often results in a truncated protein which may retain a small amount of function.
• disruptions occurring in the 5' end of the AUTS2 gene may have a lesser effect on the shorter isoform, which can then perform its normal function.
• the detection of the chromosomal abnormalities and alterations described herein may be performed on any sample from an individual which comprises nucleic acid, in particular genomic DNA. Suitable samples include bodily fluids (e.g., blood, urine, serum, cerebral spinal fluid, amniotic fluid), cells (e.g., brain cells, white blood cells), or body tissue. Standard methods are known for extracting DNA from samples.
• Nucleic acid may be used in a purified or unpurified form.
• a number of methods for detecting chromosome abnormalities are known in the art, including karyotyping, FISH, array-CGH, MAPH, sequencing assays, quantitative PCR (q-PCR), QMPSF (Quantitative Multiplex PCR of Short Fluorescent Fragments, see Casilli et all, 2001 Hum Mutat 20:218-26) and MLPA. (see U.S. Publication US2011/0281759)
• Karyotyping is a conventional procedure for determining the chromosome complement of an individual, defined both by the number and morphology of the chromosomes. Stains are used to produce banding patterns on the chromosomes which permits the identification of the chromosome and the visualization of structural abberations.
• GTG banding (G-bands by trypsin using Giemsa) is a standard cytogenetic technique.
• Hybridization assays such as fluorescence in situ hybridization (FISH) are also common techniques for detecting chromosomal abnormalities.
• FISH fluorescence in situ hybridization
• chromosomes are hybridized with "painting probes" labeled by combinations of different fluorophores. Details of this technique and variations of the techniqe are found, e.g., in Liehr et al. Histol Histopathol 2004 19:229-237.
• Array comparative genomic hybridization (array-CGH) and Multiplex Amplifiable Probe Hybridization (MAPH) are both molecular-cytogenetic methods for the analysis of copy number variation (Kousoulidou et al. Methods Mol Biol 2010 653:47-71).
• Copy number variation refers to the number of copies of a particular gene.
• Amplification assays are also known to the skilled person and are suitable for detecting chromosome abnormalities.
• 3C-qPCR is one exemplary technique (Hagege et al. 2007 Nature Protoc 2" 1722-33).
• a chromosomal abnormality in the AUTS2 gene is detected using MLPA.
• MLPA is a technique to determine copy number (Schouten et al. 2002 Nucleic Acids Res. 30:e57).
• MLPA probes are designed to hybridize to a region of interest.
• MLPA probes comprise a set of two probes. Each probe of the probe set is designed to hybridize to the target DNA in close proximity to each other.
• Each probe in a probe set has between 15-100 nucleic acids complementary the target sequence, preferably between 20-50 nucleotides (referred to as LHS and RHS, left and right hybridizing sequence, respectively.)
• Each probe has, in addition the LHS and RHS, a primer recognition sequence.
• LHS and RHS a primer recognition sequence
• a "stuffer" sequence may be inserted between the LHS/RHS and the primer recognition sequences.
• the stuffer sequence may be used to alter the length of the amplified probe in order to distinguish the particular probe set from other probe sets. Further details of this method are described in U.S. Patent No. 6,955,901 which is hereby incorporated by reference in its entirety.
• the MLPA technique has the advantage that small deletions and duplications can be detected and the location of the abnormality can be finely mapped to a specific region. MLPA permits the high resolution detection of abnormalities in specific gene candidates. It also is able to measure copy number changes with high quantitative power. In the case of the AUTS2 gene, these advantages are of importance as we have discovered that relatively small deletions can be pathogenic and the severity of the resulting disorder can be predicted based on the location of the abnormality. While MLPA is a well-known technique, its application in determining alterations in the AUTS2 gene is surprising as it was not known before the present disclosure that intragenic deletions of AUTS2 could lead to the phenotypes described in Table 1.
• One aspect of the disclosure provides multiplex ligation dependent probe
• MLPA amplification amplification
• “Specificity” for the AUTS2 gene refers to the ability of each probe in the set of two to bind adjacently to each other to the AUTS2 gene.
• Each probe of the set may also bind to other genomic sequences, however, for amplification to occur, the probes need to bind next to each other in order to ligate.
• the MPLA probe set is specific for a genomic region between exons 9-19 of the AUTS2 region, more preferably for the genomic region which encodes the short isoform.
• the probe sets may be designed to either the sense or anti-sense strand. While exon specific probes are generally preferred, some exons may not contain suitable sequences with which to design MLPA probes.
• MLPA probe pairs useful in the present invention include MLPA 102, MLPA 110, MLPA 118, MLPA 126, MLPA 130, MLPA 096, MLPA 096, MLPA 136, MLPA 140, MLPA144, MLPA 100, MLPA 108, MLPA 112, MLPA 116, MLPA 120, MLPA 124, MLPA 128, MLPA 132, MLPA 134, MLPA 105, MLPA 138, MLPA 107, and MLPA 136.
• Probe sequences are provided in Table 6. Table 6A provides the "left" probe and Table 6B provides the "right" probe for each probe pair.
• MLPA 102 probe refers to a pair of sequences comprising:
• MLPA probe design is described, e.g., in Pantano et al. BMC Genomics 2008 9:573, Zhi J. BMC Research Notes 2010 3: 137 and MRC-Holland B.V. provides a detailed probe design protocol at www.mlpa.com.
• One aspect of the disclosure provides antibodies specific for the AUTS2 short isoform of SEQ ID NO:2.
• “Specific for the AUTS2 short form” refers to antibodies which recognize SEQ ID NO:2, but not exons 1-8 of AUTS2.
• the antibody specific for the AUTS2 short isoform of SEQ ID NO:2 does not recognize the protein encoded by the longer transcript.
• the AUTS2 specific antibody is typically specific for a three dimensional epitope not present in the protein encoded by the longer transcript.
• the antibody specific for the AUTS2 short isoform of SEQ ID NO:2 can also be specific towards the unique 5' end of the shorter isoform.
• Such antibodies are useful to detect not only the presence of the AUTS2 short isoform, but also changes in expression level. Suitable antibodies include monoclonal and polyclonal antibodies as well as fragments containing the antigen binding domain and/or one or more
• an antibody fragment is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen binding region.
• the present invention also provides new therapeutic applications for treating AUTS2 syndrome and in particular autism.
• the disclosure demonstrates that loss of auts2 in zebrafish embryos causes microcephaly and decreases neuronal staining. This phenotype is rescued by the C-terminal AUTS2 human mRNA. The AUTS2 short isoform is therefore sufficient for rescuing the loss of AUTS2 in zebrafish and is useful in treating disorders mediated by AUTS2
• the disclosure provides a polypeptide comprising the AUTS2 sequence having at least 80, at least 85, preferably at least 90, more preferably at least 95% identical to SEQ ID NO:2.
• polypeptide may include additional sequences, such as protein tags (His, Flag, Myc, HA, GST, V5, GFP, etc.), it does not comprise additional AUTS2 sequences, namely from exons 1-8.
• additional AUTS2 sequences namely from exons 1-8.
• the N-terminus of said polypeptide is encoded by exon 9 of the AUTS2 gene.
• said polypeptide may include additional sequences, such as protein tags (His, Flag, Myc, HA, GST, V5, GFP, etc.), it does not comprise additional AUTS2 sequences, namely from exons 1-8.
• the N-terminus of said polypeptide is encoded by exon 9 of the AUTS2 gene.
• polypeptide comprises the alternatively spliced amino acids VRTPGRN.
• the disclosure provides a nucleic acid molecule encoding the polypeptides disclosed herein.
• the nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 80, at least 85, preferably at least 90, more preferably at least 95% identical to SEQ ID NO:2.
• the nucleic acid molecule comprises SEQ ID NO: l or a nucleic acid molecule at least 80, at least 85, preferably at least 90, more preferably at least 95% identical to SEQ ID NO: 1.
• the nucleic acid molecule may comprise additional sequences, for example, promoter or expression sequences, but does not comprise additional sequences of AUTS2, e.g., exons 1-8.
• the nucleic acid encodes a polypeptide encoded by exon 9 of the AUTS2 gene.
• the nucleic acid molecule may be provided in a vector.
• a "vector” is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached.
• the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
• Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.
• Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors, as set forth in greater detail below.
• Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.). Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided.
• the method of introduction is largely dictated by the targeted cell type include, e.g., CaP04 precipitation, liposome fusion, lipofectin, electroporation, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, viral infection, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei.
• the nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).
• AUTS2 short isoform polypeptides as described herein may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a dominant negative polypeptide.
• host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc.
• said polypeptides are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems.
• Suitable cell types include tumor cells, Jurkat T cells, NIH3T3 cells, CHO, and Cos, cells.
• the cells are culturable cells and more preferred the cells are in vitro cells.
• the nucleic acid sequence encoding the AUTS2 short isoform may be provided to a cell as part of a gene delivery vehicle.
• a gene delivery vehicle is preferably a liposome or a viral gene delivery vehicle. Liposomes are well known in the art and many variants are available for gene transfer purposes.
• Various viral gene delivery are currently used to transfer genes into target cells. In the present disclosure it is preferred to use those viral vectors that do not express their own genes but only the transferred genes.
• the nucleic acid compound is preferably provided as an expression cassette wherein the expression cassette encodes a transcript comprising said compound.
• a further aspect of the present disclosure is the provision of a vector comprising a nucleic acid molecule encoding the AUTS2 short isoform for use in gene therapy.
• Such therapy is useful in the treatment of AUTS2 syndrome.
• Methods are therefore provided for treating an individual afflicted with or at risk of developing AUTS2 syndrome comprising administering to an individual in need thereof a nucleic acid molecule encoding the AUTS2 short isoform.
• the nucleic acid molecule is preferably provided in a viral vector suitable for gene therapy.
• Appropriate vectors and delivery methods are known to a skilled person and are described, e.g., in Schlachetzki et al. Neurology, Gene therapy of the brain. (2004) 62: 1275- 1281 and Richardson et al. Neurosurg Clin N Am. 2009 20(2):205-10.
• a further aspect of the disclosure provides pharmaceutical composition
• a pharmaceutically acceptable excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.
• the compound is dissolved in a solution that is compatible with the delivery method.
• the solution is a physiological salt solution.
• excipients capable of forming complexes, vesicles and/or liposomes that deliver such a compound as defined herein in a vesicle or liposome through a cell membrane.
• Suitable excipients comprise polyethylenimine (PEI) or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, ExGen 500, synthetic amphiphils (SAINT- 18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self assembly into particles that can deliver such compounds, to a cell.
• the compositions can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;
• Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
• Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention.
• the invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release.
• Actual dosage levels of the pharmaceutical preparations described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• the disclosure provides a method of treating an individual, preferably a human, in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition as disclosed herein.
• the composition is administered intracerebrally or into the spinal cord of the individual.
• the disclosure further provides non-human animals, preferably mammals, comprising nucleic acids encoding AUTS2 short isoform.
• Methods for introducing nucleic acids into animals are known to one of skill in the art and include standard transgenic techniques such as introducing said nucleic acid into an undifferentiated cell type, e.g., an embryonic stem (ES) cell.
• ES embryonic stem
• the ES cell is injected into a mammalian embryo, where it integrates into the developing embryo. Insertion of the nucleic acid construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment.
• the embryo is implanted into a foster mother for the duration of gestation.
• Transgenic animals comprise a heterologous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially in germ cells.
• chimeras or “chimeric animals” are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals.
• transgenic animals particularly animals such as mice or rats
• Methods for generating transgenic animals have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.
• the invention further provides a non-human animal, preferably a non-human vertebrate wherein the AUTS2 short isoform is knockout on at least one and preferable two of the AUTS2 chromosomes in the cell.
• a non-human animal preferably a non-human vertebrate wherein the AUTS2 short isoform is knockout on at least one and preferable two of the AUTS2 chromosomes in the cell.
• the invention provides a non-human animal, preferably a non-human vertebrate, wherein the AUTS2 short isoform is silenced, preferably by means of an siRNA specific for the AUTS short isoform.
• said non- human animal is a mammal or fish.
• to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
• verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
• an element means one element or more than one element.
• treating includes prophylactic and/or therapeutic treatments.
• prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic, (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
• an inversion (patient 20) and a translocation (patient 21) with one of the breakpoints in AUTS2 were found by conventional karyotyping from two different laboratories; detailed genotypic and phenotypic data were available for both of these patients. In total 25 patients from 21 unrelated families were included in our analyses (see figure 1, figure 6, and table 7).
• the deletion involved a downstream gene, WBSCR17, while in patients 17 and 19 two downstream genes, WBSCR17 and CALN1 were also included in the deletion (see figure 1).
• probands had intellectual disability or a developmental delay (patient 1, 2, 4-21); only patient 3 had normal intellectual development.
• Two of the parents carrying an AUTS2 deletion had a normal intelligence (the mother of patient 3 and the father of patient 5), one had experienced learning difficulties (the mother of patient 8) and one had mild intellectual disability (mother of patient 10 and her sibling).
• Ten probands were diagnosed with AS or showed autistic behavior (patient 3, 4, 7, 9, 13, 16, 17, 20 and 21). The mother of patient 3 reported herself to have
• the phenotype caused by the observed AUTS2 aberrations is variable.
• microcephaly is strongly associated with ASD (Shinawi, M. et al. J Med Genet 47, 332-41 (2010)). Likewise, microcephaly is the most representative clinical feature amongst our patients with an AUTS2 deletion (16/43).
• sb-MOs splice-blocking morpholinos
• Table 1 Phenotypic features characterizing the AUTS2 syndrome patients
• Table S2 Control cohorts. The control cohort tested for CNV's in AUTS2 with the number of controls per cohort and the references that give more information on these cohorts. The number of deletions identified in the controls at the AUTS2 locus is indicated. Datasets 1, 2, 4, and 5 were determined with Affymetrix 6.0; dataset 3 was determined with Illumina 1M; dataset 6 was determined with Illumina 240K-650K; and dataset 7 was determined with agillent 105K/180K.
• Table S4 Overview of the location of the probes used for MLPA analysis. All features are sorted for the start position with numbering according to genome build HG18.
• Table S5 FISH probes used for breakpoint mapping on chromosome 7 and chromosome 22. Showing probe name, chromosome band, start position and end position according to the GRCh37 genome assembly.
• exon 18 L107_ _auts2 gggttccctaagggttggaTGTCTCAGTCGCAGTTATACATCAGCACGTC
• R104_auts2 /5Phos/GAACATGACAGCCAGGATGCAGGGCCGATTtctagattggatcttgctggcgcgtcc
• R116_auts2 /5Phos/TTACCATGCTACACATTGAATGTAACTGCTTTGCCtctagattggatcttgctggcgcgtcc
• R120_auts2 /5Phos/ATGGCATCAAACTGAGATTACGTGGCTTGCTCAtctagattggatcttgctggcgcgtcc
• R124_auts2 /5Phos/GTCCCACACACTTTACTCCAAAAGGACCCGAGGGTACGTGCAAAGTCtctagattggatcttgctggcgcgt.cc
• R128_auts2 /5Phos/CCAGTTAGTAGACAGCTTTAAAAGCCAGGGGTCTGAAGGCCATTtctagattggatcttgctggcgcgtcc
• R132_auts2 /5Phos/AATGTCTTCCTCCTAACCACGTTGCTCTTTCTTGTTCCAGAAACCAGGGAAGTGtctagattggatcttgctggcgcgtcc
• R136_auts2 /5Phos/CCGACCCTAGTAACAAAAGTCTCTCAGTTACCCAGGGCACattatcaggagcatttctagattggatcttgctggcgcgtcc
• R140_auts2 /5Phos/TCCAGGCTACATGAGACATGCAGACATGCTCCAGTGTCATGAACCTCCattatcaggtctagattggatcttgctggcgcgtcc
• R144_auts2 /5Phos/AGTCCAGCCGTTGTCCTAAGGAGGGAGCAGGCCACCTCAGACAGAAAATGCAGCTGGAGtctagattggatcttgctggcgcgtcc
• R102_auts2 /5Phos/CCTCGiGCAGAAGAGGACATCATTGATGGatttctagattggatcttgctggcgcgtcc
• R118_auts2 /5Phos/ATCCAAGGGCTTCCACCGGAGCAGCTCatatttctagattggatcttgctggcgcgtcc
• R126_auts2 /5Phos/CTGGCCATGTAAGGAGATACAGGGAACgattatcaggtattctagattggatcttgctggcgcgtcc
• R130_auts2 /5Phos/GCACCTTGCTGGACTGACCTGAGGAAGAGCttgaatgcgagttattatctctagattggatcttgctggcgcgtcc
• R134_auts2 /5Phos/TGTTCCCCTTGCTGTGTAGAAACAGATGCAGTCAGACCCACATAAGCTtctagattggatcttgctggcgcgtcc
• R105_auts2 /5Phos/GTTTCAGTACATCCCGTCCAGCTTCCAGATTctctagattggatcttgctggcgcgtcc
• R138_auts2 /5Phos/CTACATTCACAGGCCTAGCAGCAGTTGGTGGCAATGCCTTCGGGGGACTTGGAAATCCTTCCtctagattggatcttgctggcgcgtcc
• R136_auts2 /5Phos/CGTTTCCTGATGTGACGCACTGTATTCCAGCTGGTGATCAAGTCTGGGAAtctagattggatcttgctggcgcgtcc
• NimbleGen 135k custom (custom design 135k (custom design signature) signature) design signature
• Brain disorder ND (assymetric) ND cerebral palsy/spasticity +(assymetric)
• Huang 2 CA M09-0400 DGAP201 1
• Routine diagnostic array CGH and karyotyping was performed for intellectual disability (ID)/ multiple congenital anomalies (MCA) for a total of about 50,000 patients in ten diagnostic centers in the Netherlands, Belgium, Great Britain, the USA and Canada.
• ID intellectual disability
• MCA multiple congenital anomalies
• Peripheral blood samples of patients with an AUTS2 aberration were obtained for further studies after informed consent of the patient or parents where appropriate. Clinical information was collected through the referring physicians and with approval of the local medical ethical committee. Informed consent was obtained from parents for publishing photographs. Patients were included in this study when detailed phenotypic an genotypic data were available.
• the clinical data of patients and carrier parents were used to identify features that occurred in at least two unrelated patients. These features were added to the list of clinical features characterizing the AUTS2 syndrome for which all patients were scored positive or negative. Asymmetrically occurring features were not counted as positive. The sum of positive features was counted for each patient and was defined as the AUTS2 syndrome severity score, which was used to investigate a genotype- phenotype correlation.
• Chromosome analysis was performed on standard synchronized cultures of peripheral blood lymphocytes. In general GTG-banded chromosomes were analyzed at the 550- band level from five metaphases and the chromosomes were counted from ten metaphases. Array CGH
• the relative DNA copy numbers at the SNP/CNV loci from the Affymetrix platforms were determined by comparison of the normalized array signal intensity data for the proband's DNA sample against the HapMap270 reference file provided by Affymetrix, using Genotyping Console or ChAS software (default settings). All rare CNV's are checked using Nexus 20 . Analysis and intra-chip normalization of the Illumina image files was performed using lllumina's GenomeStudio Genotyping Module software v.2010.3 with default parameters.
• Genotype calls were generated using the Illumina-provided genotype cluster definitions file (HumanOmni2.5-4vl_B.egt, generated using HapMap project DNA samples). CNV analysis was performed using a multi- algorithm approach, as described elsewhere 25 . Data analysis of the NimbleGen arrays was performed as described previously 26 .
• Probes were designed for exons 1 to 19 of the reference sequence oiAUTS2 (supplementary table S4). Oligonucleotides were synthesized by Integrated DNA Technologies (Leuven, Belgium); all other reagents were from MRC- Holland (Amsterdam, The Netherlands). The samples were separated on a 3730 automated sequencer (ABI Systems) and analyzed using Gene marker vl.95 software (Soft genetics).
• DNA from patient 20 was sequenced using a customized whole-genome large insert jumping library for Illumina HiSeq2000 sequencing based on the mate-pair methods of Applied Biosystems SOLiD sequencing (Applied
• Biosystems Inc 23 > 24 .
• the method sequences the ends of fragments separated by large inserts ( ⁇ 3kb in this case) of genomic sequence, yielding very high physical coverage of the genome by mapping the insert between paired-end reads.
• 20 ⁇ g of DNA was sheared and size selected around 3.5 kb, cap adaptors with EcoP15I restriction sites were ligated to the ends, and fragments were circularized with an internal oligonucleotide adaptor containing a subject- specific barcode and a single biotinylated thymine.
• BAM files were processed to identify rearrangement breakpoints by a freely available C++ program, BamStat (http://mappingtools.chgr.org), we developed to tabulate mapping statistics and output lists of anomalous read-pairs (defined as having ends that map to two different chromosomes, an abnormal insert size, or unexpected strand orientations).
• the breakpoint positions were converted to the GRCh36/hgl8 genome built to allow comparison with other patients in this article. Complete details of all protocols and bioinformatic analysis are available 23 .
• FISH analysis was used for translocation breakpoint mapping in patient 21.
• Locus specific FISH probes for chromosome 7qll.22 and 22ql2.1 were selected using the Ensembl and UCSC genome browser database (http://www.ensembl.org,
• RNA rescue experiments the human wild type mRNAs, full-length and short isoform, were cloned into the pCS2 vector and transcribed in vitro using the SP6 Message Machine kit (Ambion). All the
• Proband one is born prematurely at 34 weeks of gestation and had a birth weight of 1361 gram ( ⁇ p3). His mother has used alcohol in this pregnancy and is mildly intellectual disabled. His father is diagnosed with schizophrenia. The little boy (now six years and 4 months old) started talking at 2,5 years of age. He is mildly
• Proband two is a five years and eight months old girl. She is born at term with a normal birth weight. She has a delayed psychomotor development and epilepsy. She had gastro-oesophageal reflux. Physical examination at the age of 5 years revealed borderline short stature (height at p3), microcephaly (headcircumference 45.5cm, ⁇ p2), a nasal deromid sinus and left sided muscle weakness and right sided spasticity. She is said to have soft cleft palate, but this was not objectified. A MRI of the brain was performed, which revealed colpocephaly, corpus callosum agenesis,
• hypoplasia of the brain stem and an asymmetry of the cerebral hemispheres with a smaller hemisphere and an enlarged ventricle at the right.
• ophthalmologic evaluation a tear duct hypoplasia and right sided hypoplastic optic nerve were noticed. She has amblyopia and exotropia of the right eye.
• the proband in family three is a five years and three months old boy that is the first child of non-consanguineous parents. His mother used alcohol in the first two months and antidepressants in all months of this pregnancy. He was born 5,5 weeks early. He had a birth weight of 2353 grams (p50). He has aggressive and autistic behavior. The best fitting diagnose would be pervasive developmental delay with suspicion to progress to Asperger syndrome. Physical examination at the age of 5 years and 3 months revealed a height of 106 cm (p50), a head circumference of 51,5cm (p50) and a weight of 18,5 kg (p3- 10). He has short palpebral fissures, a low nasal bridge with a bulbous nasal tip and a highly arched palate. His neck is short, and he has a shawl scrotum. Examination of his extremities was remarkable for a clinodactyly of his little finger, lax ligaments, fat pad on the pointed finger and an inversion of the foot.
• Echocardiogram revealed a small patent formamen ovale that closed spontaneously.
• Microarray revealed two variants: a deletion in intron 2 of the AUTS2 gene (chr7:69134595-69202305) and a duplication of Xp ll. l (FAAH2 and ZXDA, chrX:57356516-57658457).
• the AUTS2 deletion was confirmed by FISH and high density array (Roche Nimblegen 135K, high density custom designed by... .) ⁇ Both variants were found to be inherited from his mother, who thinks that she herself has Asperger syndrome characteristics.
• Proband four is the first child of non-consanguineous parents. He was born at term after a normal pregnancy and delivery. Anthropometric measurements at birth were within normal limits. His early developmental milestones were normal. At the age of three years he was noticed to present significant opposite behavior. Now at the age of eight years he has been diagnosed with an autism spectrum disorder (ASD). He is quiet and needs a well structured environment, but overall his communication skills are better than those of his younger brother who has also been diagnosed with ASD. He receives special education, because of his autistic behavior. His intellectual development is delayed, with a mild discrepancy between performal and verbal skills.
• ASSD autism spectrum disorder
• Array CGH (Agilent 180K) analysis revealed a de novo intragenic deletion in the AUTS2 gene. No other aberrations were noted. The deletion spans 187 to 242 kb in intron 4 of the largest A UTS2 transcript (and deletes exon 5 of the shorter alternative transcript: AUTS2-003
• This proband is the third child of non-consanguineous healthy parents.
• Both parents have no intellectual deficits nor autistic features and followed normal education. Pregnancy and delivery were uneventful. He was born at a gestational age of 41 weeks and 2 days with a birth weight of 4170 gram (p85). Early developmental milestones were mildly delayed. He started walking at the age of 18 months. At the age of 28 months he had a speech development comparable with a 16 month old child. There are (no) features of autism. Now at the age of 36 months, he is functioning at a developmental age of 23 months. Physical examination revealed a height 100 cm (p25) and a head circumference of 50.8 cm (p50). No dysmorphic features are evident (see figure 2A). Physical examination of the father showed no dysmorphic features and normal body proportions. Genetic analysis
• Array-CGH using a Lowlands consortium custom designed Agilent 105K platform, revealed an intragenic deletion of 150 kb with breakpoints at 68,900,000 (Probe A16_P17972091) and 69,050,000 (Probe A16_P17972516) removing exon 2 of the AUTS2-gene. This deletion leaves the reading frame in tact and causes a deletion of 71 amino acids. The deletion was inherited from his normal farther, who inherited the deletion from his normal mother/father.
• Patient six is a three year old girl. She is the second child of healthy non- consanguineous parents. The family history is unremarkable. She is born at term after an uneventful pregnancy and delivery. She has a delay in motor and speech development. She could sit up at the age of 10 months and started walking at 18 months of age. She received early intervention services including speech therapy and physical therapy, but has made little progress. Currently, her behavior has been most concerning as she has severe temper tantrums which might be related to her limited ability to express herself. Physical examination at the age of three years revealed a head circumference of 48.5 cm (p25). Height and weight measurements are not available. No dysmorphic features except for a left eye ptosis.
• Brain MM has been performed and showed, by report, diffuse symmetric white matter abnormality in the periventricular region with some subcortical involvement.
• Proband 7 is an 2 year and 6 months old boy. He is the second child of healthy non consanguineous parents. His older brother was diagnosed with pervasive
• This proband is the second child of non-consanguineous parents.
• the mother of this girl was born with a non-midline upper cleft lip and although she was said to have had normal early childhood development, she struggled at mainstream school with reading and writing and needed extra help.
• Antenatally, cleft lip and probable cleft palate was detected with ultrasound. She was born at 42 +2 weeks by normal vaginal delivery with birth weight 3.3 kg (p25-50). At birth, the cleft lip and palate were confirmed and a heart murmur was detected. Echocardiography revealed a atrial septal defect with left to right flow.
• the developmental milestones were delayed with smiling at 4.4 months, sitting up unsupported at 10.5 months, and walking at 22 months. Her speech was also delayed for which she was referred to speech and language therapy.
• Dysmorphic features were noted consisting of plagiocephaly, brachycephaly, facial asymmetry with the right eyes appearing larger than the left (which improved with age), left sided unilateral cleft lip repair, prominent forehead with thin lips, a relatively flattened nasal tip, short upslanting palpebral fissures and proptosis, overlapping 2 nd , 3 rd and 4 th toes, capillary haemangioma over the occipital region of the head and over the sacrum.
• Heart sounds confirmed the presence of a quiet mid- systolic murmur over the left sternal edge.
• Her mother had evidence of her cleft lip repair, ptosis, retrognatia and a narrow mouth.
• Array analysis performed using the Affymetrix 2.7M array showed an intragenic AUTS2 deletion.
• the deletion spans 52 kb and deletes exon 3 and 4 of the AUTS2 gene (chr7:69,211,000-69,263,000).
• Q-PCR analysis the deletion was shown to be inherited from her mother. Mosaicism in the mother cannot be excluded. We are awaiting samples to arrive from the maternal grandparents for further analysis.
• Patient 9 de novo deletion overlapping exon 1-4 of the AUTS2 gene, no other genes involved
• Proband nine is born at term. She is the first child of healthy non-consanguineous parents. Family history reveals no relevant diseases, birth defects or developmental problems. Pregnancy and delivery were unremarkable. Her birth weight was: 3090g (pl0-p25), and her head circumference at birth was 32 cm (p3). Her early motor milestones were obtained at a normal age; she was rolling over at six months, sitting alone at seven months and crawling at eight to nine months. Around the age of 15 months, however, a delay in development and signs of autism were noted. She was tested to have a developmental level of 5-9 months in most areas as well as decreased eye contact and was given the diagnosis autism. She has a history of low muscle tone and feeding problems, including reflux and constipation. She is sensitive to loud sounds.
• Neurological evaluation revealed normal muscle tone. A MM scan of the brain (without contrast) was normal.
• Array CGH (Signature OS Chip) revealed a de novo microdeletion of 2.3 Mb at 7ql l.22, overlapping exon 1-4 of the AUTS2 gene (chr7:67056711-69323591). The deletion is not overlapping any other genes and was confirmed by FISH and custom high density array.
• the proband in this family is a little girl of now six years of age that is the second born child of non-consanguineous parents.
• the mother has had learning difficulties and feeding difficulties. She has short stature and mild dysmorphic features.
• the pregnancy of the proband was complicated by intrauterine growth retardation and polyhydramniom. She is born by normal vaginal delivery. Her birth weight was 2.38 kgs (p2-9).
• Her height and head circumference were below the 0.4 th centile. She is hyperteloric, has a ptosis and a downslant of the palpebral fissures, a mild proptosis and has highly arched eyebrows. She has a broad nasal bridge, a bulbous nasal tip, simple ears, low columella, strabismus and retrognatia. Her older brother, now ten years old, had a similar antenatal and postnatal course. His birth weight was 2.26 kg (p0.4-2). He fed poorly and failed to thrive with difficulty weaning onto solids. His early developmental milestones were delayed. He smiled at four to five months, crawled at 18 months and walked at two years.
• Proband eleven is a 5,5 year old boy. He is born after an uneventful pregnancy and had a birth weight of (p ). He did not begin to talk until he was 3 years of age. He is hyperactive and shows a global development delay. He is now able to talk in three to four word sentences. Physical examination at the age of 5 years and 6 months reveals a short stature (height: 106.5 cm, p lO) a weight of 14.7 kg (p25) and microcephaly (head circumference: 45.5 cm «p2, p50 for a 9-month-old).
• Chromosome analysis revealed a normal male karyogram: 46, XY. Methylation testing for Angelman syndrome was normal and Fragile X DNA testing was normal too.
• Array CGH Alent 105K identified an intragenic AUTS2 deletion (chr7:69612584- 69827896) deleting exon 6. This deletion causes the AUTS2 sequence to go out frame from exon 7 onwards. It is unknown if the deletion occurred de novo as the parents are not yet tested.
• Proband 12 was born at term and has healthy non-consanguineous parents.
• hyperteloric has epicanthal folds and micrognatia. His ears are large and low set, and he has large hands and feet. Neurologic examination was remarkable for increased deep tendon reflexes and hypertonia of both legs. A MM of the brain was normal.
• Array CGH (Roche NimbleGen 135k, a Signature Genetics custom design) was performed and showed a small intragenic deletion of the AUTS2 gene involving exon 6 (chr7:69654259-69828289). This deletion causes the AUTS2 sequence to go out frame from exon 7 onwards. The deletion was visualized by FISH and confirmed by high density array (custom design Roche Nimblgen 135K). Both parents of the boy tested negative for the deletion, which implies the deletion to be de novo.
• Proband 13 is the first child of non-consanguineous parents. She was born at a gestational age of 42 weeks after an uneventful pregnancy, but had a slow start (with an apgar score of 5 after 1 minute). Blood gas parameters were normalised one hour after birth. She had a birth weight of 3690 gram (p55). In the neonatal period she was tachypnoeic, hypotonic and inactive. In the first months of her life she had feeding difficulties, failure to thrive and needed tube feeding. Physical examination at that time revealed low muscle tone, an upslant of the palpebral fissures, strabism and an expressionless face. At childhood (7- 10 years) she suffered from recurrent upper airway infections.
• Proband 14 is a 2 years and 11 months old girl with developmental delay. Family history was unremarkable. She was born at term and had a birth weight of 2,55 kg ( ⁇ p3) She had an inguinal hernia, which was successfully operated at the age of 3 months. At the age of 5 months there was a choking incident for which she was evaluated in hospital. TORCH- and metabolic screen was normal, as was the EEC. At the age of 11 months an infected preauricular sinus was operated. Although autism was suspected at a younger age, during evaluation at the age of 25 months there was no clinical suspicion of autism but she had a mild global developmental delay of 3/9 months.
• Her tone and reflexes were normal throughout, with only mild restriction of dorsiflexion in left ankle remaining. Brain MRI at 13 months showed nonspecific hyper intense signal changes in the white matter in the periventricular zones at fron to-parietal areas. The lateral and third ventricles were prominent. MRS and MRA were normal.
• Rett syndrome testing (by sequencing of MeCP2) was negative.
• Array analysis using Affymetrix SNP array 6.0 showed a deletion of 7qll.22.
• the minimal deletion spans 330 kb of the AUTS2 gene and includes exons 6- 11 (chr7:69,544,241-69,874,867).
• the deletion was confirmed to be de novo by Q-PCR and FISH of the proband and her parents.
• the 15th patient is an 1 year and 11 months old girl with a mild developmental delay. She was born at term and had a normal birth weight. Her medical history was remarkable for a metopic suture stenosis. She is mildly delayed in general
• microcephaly with a head circumference of 44 cm, ( ⁇ p l). She had some mild dysmorphic features, namely elevated eyebrows, proptosis and epicanthal folds.
• Array CGH (Agilent 105k custom designed by Signature Genetics) revealed an intragenic microdeletion in AUTS2, deleting at least exon 6 (chr7:69699592- 69828036). The deletion was confirmed by FISH and High density Array (custom design Roche Nimblgen 135K). With High density array it was clarified that the deletion contains exon 6 to 18 of the AUTS2 gene.
• Patient 16 (deletion exon 7-19, de novo):
• Proband 16 is a 7 years and 2 months old girl. This little girl was born premature. In the neonatal period an intraventricular hemorrhage was noticed. She had some feeding difficulties and development was delayed. She has had one febrile seizure, but no signs of epilepsy at this moment. At the age of 6 years she was almost non-verbal, using some signs and communication aids. She was functioning at a developmental level of a 2.5 years old child. She meets the criteria for PDD-NOS. She is a picky eater and just stays inside the normal range with her weight with a lot of effort from her mom. Physical examination at the age of 6 years revealed a height of 110.7 cm (p20), her weight was 17.5 kg (plO), and her head circumference 50 cm (p50).
• Metabolic screening revealed an elevated C5-DC acylcarnitine, which suggests the diagnosis of glutaric aciduria type I (GA1). This was however excluded with DNA testing of the Glutaryl Co-A Dehydrogenase gene. MRI of the brain showed somewhat large ventricles and a mammillary body atrophy. No white matter abnormalities were observed. There was no evidence of neonatal brain damage.
• Microarray was performed (using the Agilent 244K array CHG) and demonstrated a deletion of exon 7-19 of the AUTS2 gene (Chr7:69842425-69895671). Additional testing did not show this deletion in both parents, indicating that it is a de novo deletion.
• the 17th patient is a boy of now 5 years and 7 months of age. He is the child of non- consanguineous healthy parents. His mother is born premature and had Rhesus disease at birth and a developmental delay. He was born full term and had a birth weight 3520 gram (p50). Pregnancy and delivery were uneventful. He has been successfully operated for an umbilical and an inguinal hernia. A g-tube was placed, because of significant failure to thrive. The developmental milestones were
• Cardiovascular examination revealed a murmur that appeared to be functional in nature and a hemodynamically stable patent foramen ovale with small left to right shunt. The boy was diagnosed with mild cerebral palsy with severe cognitive deficits. MRI results revealed a mild prominence of the ventricular system and extra-axial spaces but were otherwise unremarkable.
• Proband 18 is a 9 years and 1 month old girl. She was the third child of healthy non- consanguineous parents. She has two healthy siblings. Apart for seizures treated with medication in a paternal cousin there are no relevant disorders in the family. The pregnancy was uncomplicated except for small fetal size. At birth, she weighted 2523 gram and was determined to be small for gestational age. Weight gain was followed closely and she did well. She has a developmental delay with relative strength in gross motor skills. She has a history of hypersensitivity to loud sounds. Physical examination reveals a head circumference of 50.5 cm (p25). Her height was 131 cm and weight was 27.3 kg, both at the 30th percentile for age. She has a large tongue and tends to hold her mouth open.
• She has a flat nasal bridge, incurving 5th finger on the left and very small fingernails. She shows asymmetric developmental posturing on stressed gaits (left-sided). Muscle and strength were normal. Heel cords were slightly tight but both ankles could be flexed to neutral position. Tone throughout was otherwise within normal limits. Deep tendon reflexes were 2+ throughout and symmetrical. Toes were down-going.
• Array CGH (Agilent 244K) revealed a deletion of 2.4 mb, deleting AUTS2 in total and part of WBSCR17 (chr7:67, 771,230-70,363,865). Parental analysis revealed the deletion to be de novo. (This patient was submitted to the Decipher database, https://decipher.sanger.ac.uk/patient/251781, 9 November 2011) Patient 19 (de novo deletion of the complete AUTS2 gene and the WBSCR17 and CALN1 genes)
• Patient 19 involves a little boy of now 4 years old. He was born at term after a normal pregnancy and delivery and had a good start. His birth weight was 3658 gram (p50). Because of severe feeding problems he was admitted to the special care baby unit. He needed tube feeding. He was then discovered to have a diaphragm eventration for which he was operated when he was two weeks old. Cardiologic evaluation revealed a atrium septum defect. Further more distal hypospadias was noticed. Because of severe feeding problems a percutaneous endoscopic gastrostomy (PEG) tube was placed. On physical examination at the age of 21 months his height is 81 cm (p9), his weight 11, 1 kg (p9-25) and his head circumference 47,6 (p2). He is slightly
• hyperteloric has short upslanting palpebral fissures, a broad nasal bridge, a short philtrum, a narrow mouth and a micrognatia. He is hypotonic. Evaluation at the age of two years revealed that he is still struggling with his feeding and is still being fed through his PEG tube. He seems to be delayed in motor and speech development. He was cruising but not walking yet and he had about 10-15 words. He is sensitive to loud sounds. He was tip toeing especially on the left and an increased tone around his left ankle and slightly brisk reflexes were noted at that time. Further neurologic examination was unremarkable. During re-evaluation at three years of age he showed improvement. He does however show some evidence of dystonic stiffness of his left ankle. Dysmorphic features were more or less unchanged. An MM of the brain showed no abnormalities.
• Neurological evaluation the reflexes are present at knees, but hard to obtain in upper limbs and ankles. She has mild dysmorphic features, namely: short palpebral fissures with a mild upslant and a short philtrum and narrow mouth. Genetic analysis:
• Chromosomal analysis revealed a paracentric inversion of chromosome 7
• the 21st proband is the first child of non-consanguineous healthy parents.
• the pregnancy and delivery were uneventful. She was born at 37 weeks of gestation and had Apgar scores of 9 and 10 after 1 and 5 minutes, respectively.
• birth weight was 2740 grams (p30). Due to hypoglycemia and feeding problems she was admitted in the neonatal intensive care unit for three weeks. Feeding remained difficult during the first months after birth. At the age of five months she had an axial hypotonia and a failure to thrive. Physical examination at that age revealed mild dysmorphic features; low-implanted ears, highly arched eyebrows, a broad nasal bridge, hypertelorism, short palpebral fissures and epicanthic folds and a full lower lip (see figure 2D).
• chromosomes a systematic series of FISH analyses was performed. On chromosome 7, the clones RP11-689B18 (location on chr7:69996264- 70178753, including exon 6) map proximally and RP11-575M4 (location on chr7: 70185711-70372702, including exons 7-19) spans the breakpoint.
• G248P84061D2 all map distally to the breakpoint, clearly indicating that the translocation interrupts the AUTS2 gene, starting in intron 6 of the gene.
• clones RP11-699H18 and RP11-772E17 flank the breakpoint, while clone RP11-1056M20 spans the breakpoint (table S4).
• Ref ID 94 Livak,K.J. & Schmittgen,T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method.

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