Erwin Petek

Disruption of a Novel Gene (IMMP2L) by a Breakpoint in 7q31 Associated with Tourette Syndrome

Gilles de la Tourette syndrome (GTS) is a complex neuropsychiatric disorder characterized by multiple motor and phonic tics. We identified a male patient with GTS and other anomalies. It was determined that he carried a de novo duplication of the long arm of chromosome 7 [46,XY,dup(7)(q22.1-q31.1)]. Further molecular analysis revealed that the duplication was inverted. The distal chromosomal breakpoint occurred between the two genetic markers D7S515 and D7S522, which define a region previously shown to be disrupted in a familiar case of GTS. Yeast and bacterial artificial chromosome clones spanning the breakpoints were identified by means of FISH analysis. To further characterize the distal breakpoint for a role in GTS, we performed Southern blot hybridization analysis and identified a 6.5-kb SacI junction fragment in the patients genomic DNA. The DNA sequence of this fragment revealed two different breaks in 7q31 within a region of approximately 500 kb. IMMP2L, a novel gene coding for the apparent human homologue of the yeast mitochondrial inner membrane peptidase subunit 2, was found to be disrupted by both the breakpoint in the duplicated fragment and the insertion site in 7q31. The cDNA of the human IMMP2L gene was cloned, and analysis of the complete 1,522-bp transcript revealed that it encompassed six exons spanning 860 kb. The possible role of IMMP2L and several other candidate genes within the region of chromosomal rearrangement, including NRCAM, Leu-Rch Rep, and Reelin, is discussed. The 7q31 breakpoint interval has also been implicated in other neuropsychiatric diseases that demonstrate some clinical overlap with GTS, including autism and speech-language disorder.

An X-Linked Myopathy with Postural Muscle Atrophy and Generalized Hypertrophy, Termed XMPMA, Is Caused by Mutations in FHL1

We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.

Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

The human γ-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression

Abstract Terminal deletions of 1p36 result in a specific and common syndrome characterised by the following: growth delay, distinctive facial anomalies, hearing and visual deficits, heart defects, body asymmetry, moderate to severe psychomotor retardation, epilepsy, and self-abusive behaviour. The human gamma-aminobutyric acid A receptor delta-subunit gene ( GABRD ) encodes for one of at least 15 ligand-gated chloride channels for gamma-aminobutyric acid ( GABA ), the major inhibitory neurotransmitter in the mammalian brain. Recently we have mapped this gene by radiation hybrid mapping to the critical region of gene loss of the 1p36 deletion syndrome within 1p36.33. The complete complementary DNA (cDNA) sequence of GABRD was generated using assembled sequence of cDNA fragments already available, and 5′-rapid amplification of cDNA ends products. Fine physical mapping of the GABRD gene within this genomic interval was performed by screening bacterial artificial chromosome contigs spanning the critical region of the 1p36 deletion syndrome. The GABRD gene maps immediately proximal to the PRKCZ gene that is located between marker D1S243 and cosmid D1Z2 – a region thought to be critical for cognition and speech development. The GABRD gene is expressed most abundantly in brain and has three alternative exons (1A–C) with alternative start codons at the 5′-end. Genomic localisation, function, and expression would suggest that the GABRD gene represents a good candidate for the neurodevelopmental and neuropsychiatric anomalies seen in the 1p36 deletion syndrome.

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.

Identification of risk genes for autism spectrum disorder through copy number variation analysis in Austrian families

Autism or autism spectrum disorder (ASD) is a range of neurodevelopmental disorders starting in early childhood and is characterized by impairments in communication and reciprocal social interaction and presence of restricted and repetitive patterns of behavior. The contribution of genetic factors to autism is clear in twin and family studies. It is apparent that, overall, ASD is a complex non-Mendelian disorder. Recent studies suggest that copy number variations (CNVs) play a significant role in the etiology of ASD. For the current work, we recruited 245 family members from 73 ASD families from Styria, Austria. The DNA from probands was genotyped with Affymetrix single nucleotide polymorphism (SNP) 6.0 microarrays to screen for CNVs in their genomes. Analysis of the microarray data was performed using three different algorithms, and a list of stringent calls was compared to existing CNV data from over 2,357 controls of European ancestry. For stringent calls not present in controls, quantitative real-time PCR (qRT-PCR) was used to validate the CNVs in the probands and in their family members. Twenty-two CNVs were validated from this set (five of which are apparently de novo), many of which appear likely to disrupt genes that may be considered as good candidates for neuropsychiatric disorders, including DLG2, S100B, ARX, DIP2A, HPCAL1, and GPHN. Several others disrupt genes that have previously been implicated in autism, such as BDNF, AUTS2, DPP6, and C18orf22, and our data add to the growing evidence of their involvement in ASD.

Molecular characterisation of a 15 Mb constitutional de novo interstitial deletion of chromosome 3p in a boy with developmental delay and congenital anomalies

AbstractWe describe the case of a 22-month-old boy with developmental and psychomotor retardation as well as craniofacial dysmorphism, including a cleft lip. Analysis of G-banded chromosomes of the propositus showed a de novo interstitial deletion of the short arm of chromosome 3, del(3)(p13p11). Fine mapping of the deletion was performed using fluorescence in situ hybridisation analysis with region-specific BAC clones. Eight BACs were absent from one chromosome 3 from the patient. Molecular analyses of eleven polymorphic DNA markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 3 is of paternal origin. The deleted segment encompasses about 15 Mb between marker D3S3551 and the centromere. Only a small number of known genes, including PROK2, GPR27, RYBP, PPP4R2, ROBO1, and GBE1, which map in the 3p13-p11 region are included in the deletion.

Molecular characterisation of a 3.5 Mb interstitial 14q deletion in a child with several phenotypic anomalies

Interstitial deletions of the long arm of chromosome 14 are infrequent. Molecular and clinical studies on patients with deletions involving 14q11.2-q21 have recently been reported.1 Most of these deletion patients share common clinical signs, such as midline defects of the central nervous system, feeding problems, growth abnormalities, hypotonia, developmental delay, mental retardation, and craniofacial anomalies.1 Here we present the phenotypic, cytogenetic, and molecular genetic findings of a 2½12 year old boy with a 14q12-q13.1 deletion. To our knowledge this is the second case described of a patient with a deletion of less than 3.5 Mb within chromosome bands 14q12-q13.1. Using the genomic sequence between markers D14S1060 and D14S286, we have constructed a transcription map of the genomic interval deleted in our patient. Our proband (fig 1) is the 40 week product of a second, uncomplicated pregnancy and delivery. Maternal age was 27 years. Birth weight was 4435 g (>90th centile), length 55 cm (>90th centile), and head circumference 34 cm (10th-25th centile). Apgar score was 7/9/10. The neonatal period was complicated by pneumonia, treated with parenteral antibiotic therapy over 10 days. Besides relative microcephaly, hypertelorism, epicanthic folds, a long and flat philtrum, hypodontia, laterally placed, hypoplastic mamillae, second degree hypospadias, bifid scrotum, and bilateral cryptorchidism were noticed. Cranial ultrasound was normal; ultrasound of the kidneys and pelvic region showed bilateral second degree hydronephrosis, and both testes were visible in the inguinal region. Screening for connatal infections (toxoplasmosis, rubella, cytomegalovirus, and parvovirus B19) was negative. Psychomotor development was severely impaired from early infancy. Lack of vision was evident at 3 months and, apart from head control, no gain of motor milestones or social contact was achieved until the age of 29 months. Muscle tone of the trunk was decreased while it was increased and dystonic in the upper and …

Molecular characterization of a 12q22‐q24 deletion associated with congenital deafness: Confirmation and refinement of the DFNA25 locus

The DFNA25 locus for autosomal dominant nonsyndromic hereditary hearing loss has been mapped to 12q21‐q24 by linkage analysis. A de novo deletion in a six‐year‐old boy with congenital hearing loss as well as mental and motor retardation now provides independent confirmation of this genetic localization and narrows the critical interval to 13 cM in the 12q22‐q24.1 region. Mapping of the deletion was performed using fluorescence in situ hybridization (FISH) analysis with region‐specific yeast artificial chromosome (YAC) clones. Ten YACs 929_e_4, 959_c_3, 746_h_7, 817_h_10, 886_a_6, 916_h_9, 969_c_12, 747_e_2, 812_h_12, and 959_f_8 were absent from one chromosome 12 from the patient. Molecular analyses of eight polymorphic markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 12 is of paternal origin. Several known genes including ATP2A2, UBE3B, and VR‐OAC that map in the 12q22‐q24.1 region are included in the deletion. These results provide evidence that haploinsufficiency for a gene or genes in 12q22‐q24.1 is associated with autosomal dominant deafness.

Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non‐invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low‐volume on‐chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non‐related individuals. Single‐cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non‐related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single‐cell basis.