Profile and facial view of the t(1;6) translocation patient at the age of eight years. Written permission was obtained from the child’s parents for these images to be reproduced. 

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... novo balanced chromosome rearrangements that disrupt or otherwise inactivate genes in the breakpoint regions can cause mental retardation or congenital malformations or both. 1–3 The cytogenetic and molecular characterisation of disease associated balanced chromosome rearrangements offers a successful instrument for identifying genes involved in inherited disease. Over the past decade, well over 100 genetic loci and genes of Mendelian disorders have been found by such positional cloning approaches. 4 5 Here we describe our analysis of a de novo t(1;6)(q32.3;q22.3) translocation in a dysmorphic and devel- opmentally delayed boy with an early childhood history of recurrent chest infections. The constitutional chromosome breakpoint on chromosome 6q22 is of particular interest, because it coincides with a common breakpoint cluster region in T cell malignancies. The TCBA1 gene is thought to be a target gene for the tumour specific chromosome rearrangements; however, its function remains to be elucidated. 6 Our patient provides important clues as to the phenotypic effects of constitutional TCBA1 haploinsufficiency. The patient, a boy, was clinically assessed by one of us (DTP) and recruited to the study with parental consent. He was the oldest child of healthy non-consanguineous parents. There was no history of miscarriages. He was born at 38 weeks’ gestation. His birth weight was on the 50th centile, and his occipito-frontal head circumference (OFC) was between the 75th and 91st centiles. He was found to have an atrioven- tricular septal defect, which closed spontaneously in the first few months of life, and a slightly dysplastic pulmonary valve. Macrocephaly was also noted and thought to be familial, with a paternal OFC of 5 cm above the 99.6th centile, and maternal OFC on the 91st centile. He had bilateral orchidopexies for undescended testes, and a fundoplication for marked gastro-oesophageal reflux. Other problems included recurrent chest infections, which had improved, asthma, and enuresis. His development was delayed, and formal assessment at 17 months showed a mental age of 12.3 months (GQ = 78). He had general learning difficulties, especially in the area of expressive speech. When assessed at the age of five years, his height was on the 25th centile, weight on the 75th centile, and OFC between the 75th and 91st centiles. He was very fair skinned and had very blond, almost white hair, although both his parents had darkish hair. He was hyperteloric, and had blue eyes and small palpebral fissures. He also had prominent corneal nerves. His nose was prominent and he had a thin, tented upper lip. He had fetal finger pads, overlapping third and fifth toes, very hyperextensible joints, and a slightly hirsute back. On follow up at the age of eight years his facial features had coarsened and his hair had become darker (fig 1). He had also been extensively investigated for frequent loose stools, for which so far no cause had been found. He continued to have difficulties with his motor skills, and communication, particularly in the area of expressive speech. Investigations have included cranial magnetic resonance imaging, which showed some enlargement of lateral and third ventricles with no evidence of aqueduct stenosis. A renal ultrasound, EEG, thyroid function test, sweat test, and lysosomal enzyme screen were normal. Immunological studies did not show any specific abnormalities of the lymphocyte subpopulations or of immunoglobulin and anti- body production. However, he was found to have raised antineutrophil cytoplasmic antibodies of the cytoplasmic pattern (C-ANCA), which are often associated with vasculitides. BAC and PAC clones were selected from the Wellcome Trust Sanger Institute ensembl contigs () and obtained from the resource centre primary database of the German Human Genome Project (). In order to amplify larger (approximately 10 kb) BAC/PAC subfragments, the Expand Long Template polymerase chain reaction (PCR) system (Roche Products, Basel, Switzerland) was used according to the recommendations of the manu- facturer, with a series of primer pairs (table 1) chosen from the genomic sequence of breakpoint spanning clones. Genomic BAC/PAC DNAs and their long range PCR products were labelled with biotin-16-dUTP or digoxigenin-11-dUTP (Roche) by standard nick translation. Fluorescence in situ hybridisation (FISH) was carried out on metaphase spreads from EBV transformed lymphoblastoid cells using standard molecular cytogenetic techniques. 3 Total RNA was extracted from mouse tissues or whole embryos with Trizol reagent (Invitrogen, San Diego, California, USA) and reversely transcribed using Superscript III reverse transcriptase (Invitrogen). cDNA was synthesised from 3 m g of total RNA at 55 ̊ C with mouse Tcba1 reverse transcription primer (5 9 -cgattcgcagagacttaga-3 9 ); 2 m l of this cDNA were then used as template in a PCR reaction with mouse Tcba1 forward (5 9 -atgggttattgcagtggca-3 9 ) and reverse primers. PCR was carried out with an initial denaturation at 94 ̊ C for three minutes, 30 cycles of 94 ̊ C for 45 seconds, 56 ̊ C for 45 seconds, 72 ̊ C for 1.5 minutes, and a final 10 minute extension at 72 ̊ C. To obtain the full length cDNA sequence, rapid amplification of cDNA ends (RACE) was done with the SMART RACE cDNA amplification kit (Clontech, Palo Alto, California, USA), according to the manufacturer’s instruc- tions. Briefly, 5 9 -RACE PCR was undertaken with a Tcba1 specific primer (5 9 -agcgatggtcttctggagcccagtctgg-3 9 ) and Universal Primer A mix from the kit. 3 9 -RACE was done with another gene specific primer (5 9 -gtcccccagtcacatcctt- taggcgaacac-3 9 ) and Universal Primer A mix. PCR was carried out with five cycles of 94 ̊ C for 30 seconds, 72 ̊ C for three minutes, five cycles of 94 ̊ C for 30 seconds, 70 ̊ C for 30 seconds, 72 ̊ C for three minutes, and 25 cycles of 94 ̊ C for 30 seconds, 68 ̊ C for 30 seconds, 72 ̊ C for three minutes, with a final 10 minute extension at 72 ̊ C. The PCR products were cloned into pCR2.1-TOPO vector (Invitrogen) and sequenced. DNA sequence analysis was done using the genome BLAST program ( Amino acid sequence alignment and transmembrane helix prediction were performed using the TmHMM and Clustal programs of Heidelberg Unix Sequence Analysis Resources (HUSAR) (). Chromosome banding analysis of this patient revealed an apparently balanced reciprocal translocation involving the exchange of material between the long arms of chromosomes 1 and 6 (fig 2A). The parental karyotypes were normal. In order to map the de novo translocation breakpoints at higher DNA resolution, we have assembled BAC/PAC contigs of the critical chromosome regions 1q32.3 and 6q22.3 according to the database (fig 2D). Individual BAC/PACs from the breakpoint regions were co-hybridised with chromosome 1pter and 6pter identification probes to the patient’s metaphase spreads. BAC RP11-338C15 at 209.4 Mb on chromosome 1 and PAC RP1-157N22 at 124.6 Mb on chromosome 6 produced FISH signals on the normal chromosome 1 and 6, respectively, and on both derivative chromosomes (data not shown), as expected for breakpoint spanning clones. To narrow down the breakpoint regions further, three long range PCR products—338C15A, 338C15B, 338C15D—were generated from the chromosome 1 breakpoint BAC and five PCR products—157N22A, B, C, D, and E—from the chromosome 6 breakpoint PAC (table 1). The 9.8 kb BAC fragment 338C15D produced a split hybridisation signal on the derivative chromosomes (fig 2B) and therefore must contain the 1q32.3 breakpoint. Similarly, the 6q22.3 breakpoint was localised to the 12.8 kb PAC fragment 157N22E, which hybridised to the normal chromosome 6 and both derivative chromosomes (fig 2C). According to our high resolution FISH mapping results the translocation breakpoint ...