Mapping of Deletion and Translocation Breakpoints in 1q44 Implicates the Serine/Threonine Kinase AKT3 in Postnatal Microcephaly and Agenesis of the Corpus Callosum
2007; Elsevier BV; Volume: 81; Issue: 2 Linguagem: Inglês
10.1086/519999
ISSN1537-6605
AutoresElena Boland, Jill Clayton‐Smith, Victoria G. Woo, Shane McKee, Forbes D.C. Manson, Līvija Medne, Elaine H. Zackai, Eric A. Swanson, David Fitzpatrick, Kathleen J. Millen, Elliott H. Sherr, William B. Dobyns, Graeme Black,
Tópico(s)Prenatal Screening and Diagnostics
ResumoDeletions of chromosome 1q42-q44 have been reported in a variety of developmental abnormalities of the brain, including microcephaly (MIC) and agenesis of the corpus callosum (ACC). Here, we describe detailed mapping studies of patients with unbalanced structural rearrangements of distal 1q4. These define a 3.5-Mb critical region extending from RP11-80B9 to RP11-241M7 that we hypothesize contains one or more genes that lead to MIC and ACC when present in only one functional copy. Next, mapping of a balanced reciprocal t(1;13)(q44;q32) translocation in a patient with postnatal MIC and ACC demonstrated a breakpoint within this region that is situated 20 kb upstream of AKT3, a serine-threonine kinase. The murine orthologue Akt3 is required for the developmental regulation of normal brain size and callosal development. Whereas sequencing of AKT3 in a panel of 45 patients with ACC did not demonstrate any pathogenic variations, whole-mount in situ hybridization confirmed expression of Akt3 in the developing central nervous system during mouse embryogenesis. AKT3 represents an excellent candidate for developmental human MIC and ACC, and we suggest that haploinsufficiency causes both postnatal MIC and ACC. Deletions of chromosome 1q42-q44 have been reported in a variety of developmental abnormalities of the brain, including microcephaly (MIC) and agenesis of the corpus callosum (ACC). Here, we describe detailed mapping studies of patients with unbalanced structural rearrangements of distal 1q4. These define a 3.5-Mb critical region extending from RP11-80B9 to RP11-241M7 that we hypothesize contains one or more genes that lead to MIC and ACC when present in only one functional copy. Next, mapping of a balanced reciprocal t(1;13)(q44;q32) translocation in a patient with postnatal MIC and ACC demonstrated a breakpoint within this region that is situated 20 kb upstream of AKT3, a serine-threonine kinase. The murine orthologue Akt3 is required for the developmental regulation of normal brain size and callosal development. Whereas sequencing of AKT3 in a panel of 45 patients with ACC did not demonstrate any pathogenic variations, whole-mount in situ hybridization confirmed expression of Akt3 in the developing central nervous system during mouse embryogenesis. AKT3 represents an excellent candidate for developmental human MIC and ACC, and we suggest that haploinsufficiency causes both postnatal MIC and ACC. Human geneticists have often taken advantage of structural chromosome rearrangements to map and clone human disease genes, including the causative genes for human brain and eye malformations such as holoprosencephaly (SHH, SIX3, TGIF, and ZIC2), Dandy-Walker malformation (ZIC1 and ZIC4), lissencephaly (DCX and LIS1), microphthalmia (SOX2), and anterior-chamber defects (FOXC1 and PAX6), among others.1Belloni E Muenke M Roessler E Traverso G Siegel-Bartelt J Frumkin A Mitchell HF Donis-Keller H Helms C Hing AV et al.Identification of sonic hedgehog as a candidate gene responsible for holoprosencephaly.Nat Genet. 1996; 14: 353-356Crossref PubMed Scopus (521) Google Scholar, 2Roessler E Belloni E Gaudenz K Jay P Berta P Scherer SW Tsui LC Muenke M Mutations in the human sonic hedgehog gene cause holoprosencephaly.Nat Genet. 1996; 14: 357-360Crossref PubMed Scopus (905) Google Scholar, 3Gripp KW Wotton D Edwards MC Roessler E Ades L Meinecke P Richieri-Costa A Zackai EH Massague J Muenke M et al.Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination.Nat Genet. 2000; 25: 205-208Crossref PubMed Scopus (327) Google Scholar, 4Wallis DE Roessler E Hehr U Nanni L Wiltshire T Richieri-Costa A Gillessen-Kaesbach G Zackai EH Rommens J Muenke M Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly.Nat Genet. 1999; 22: 196-198Crossref PubMed Scopus (325) Google Scholar, 5Hamilton G Brown N Oseroff V Huey B Segraves R Sudar D Kumler J Albertson D Pinkel D A large field CCD system for quantitative imaging of microarrays.Nucleic Acids Res. 2006; 34: e58Crossref PubMed Scopus (19) Google Scholar, 6Reiner O Carrozzo R Shen Y Wehnert M Faustinella F Dobyns WB Caskey CT Ledbetter DH Isolation of a Miller-Dieker lissencephaly gene containing G protein β-subunit-like repeats.Nature. 1993; 364: 717-721Crossref PubMed Scopus (860) Google Scholar, 7Chong SS Pack SD Roschke AV Tanigami A Carrozzo R Smith ACM Dobyns WB Ledbetter DH A revision of the lissencephaly and Miller-Dieker syndrome critical regions in chromosome 17p13.3.Hum Molec Genet. 1997; 6: 147-155Crossref PubMed Scopus (148) Google Scholar, 8Grinberg I Northrup H Ardinger H Prasad C Dobyns WB Millen KJ Heterozygous deletion of the linked genes ZIC1 and ZIC4 is involved in Dandy-Walker malformation.Nat Genet. 2004; 36: 1053-1055Crossref PubMed Scopus (161) Google Scholar, 9Fantes J Ragge NK Lynch SA McGill NI Collin JR Howard-Peebles PN Hayward C Vivian AJ Williamson K van Heyningen V et al.Mutations in SOX2 cause anophthalmia.Nat Genet. 2003; 33: 461-463Crossref PubMed Scopus (404) Google Scholar, 10Mears AJ Jordan T Mirzayans F Dubois S Kume T Parlee M Ritch R Koop B Kuo W-L Collins C et al.Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly.Am J Hum Genet. 1998; 63: 1316-1328Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 11Glaser T Walton DS Maas RL Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene.Nat Genet. 1992; 2: 232-239Crossref PubMed Scopus (546) Google Scholar, 12Jordan T Hanson I Zaletayev D Hodgson S Prosser J Seawright A Hastie N van Heyningen V The human PAX6 gene is mutated in two patients with aniridia.Nat Genet. 1992; 1: 328-332Crossref PubMed Scopus (456) Google Scholar, 13Gleeson JG Allen KM Fox JW Lamperti ED Berkovic S Scheffer I Cooper EC Dobyns WB Minnerath SR Ross ME et al.Doublecortin, a brain-specific gene mutated in human X-linked lissencephaly and double cortex syndrome, encodes a putative signaling protein.Cell. 1998; 92: 63-72Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar We have performed mapping studies of six patients with rearrangements of distal 1q42-q44 associated with several different brain malformations, including congenital and postnatal microcephaly (MIC), agenesis of the corpus callosum (ACC [MIM 217990]), cerebellar vermis hypoplasia (CVH), and polymicrogyria (PMG). The abnormalities of three patients with MIC, ACC, and CVH consist of two large interstitial deletions of distal 1q42- or 1q43-q44 and a complex unbalanced rearrangement with two deleted and two inverted segments between 1q41 and 1q44. Another boy with postnatal MIC and ACC but not CVH has an apparently balanced reciprocal translocation between distal 1q44 and 13q32. The remaining patient had CVH and PMG but not MIC or ACC and had a terminal 1q44 deletion resulting from unbalanced segregation of a translocation between 1q44 and 11p15. We identified two genes—AKT3 and ZNF238—closest to the chromosome 1 translocation breakpoint, performed expression studies of mouse embryos, and sequenced both genes and an intervening highly conserved noncoding sequence in a cohort of 47 patients with ACC. Our results collectively implicate haploinsufficiency of AKT3 as a cause of human MIC and ACC. We ascertained six children with structural rearrangements of distal chromosome 1q who presented at our local institutions or were referred to our research projects. Three were evaluated as newborns with complicated neonatal courses, whereas the remaining three were evaluated during early childhood because of developmental delay. All six had multiple congenital anomalies that prompted chromosome analysis. We (W.B.D. and E.H.S.) selected 55 patients with ACC and normal chromosome analysis from our large Brain Malformation Research Databases for sequencing of positional candidate genes identified in the patients with 1q4 deletion and translocation. This cohort included 11 patients with ACC only, 8 with ACC and congenital MIC (some with other brain anomalies), 10 with ACC and cerebellar hypoplasia, and 26 with ACC and other anomalies. aCGH was performed using a high-resolution BAC-tiling set from the University of California–San Francisco (UCSF) Comprehensive Cancer Center that contains ∼33,000 BAC PCR-based representations, each printed once on 18×36-mm2 chromium-coated slides.14Ishkanian AS Malloff CA Watson SK DeLeeuw RJ Chi B Coe BP Snijders A Albertson DG Pinkel D Marra MA et al.A tiling resolution DNA microarray with complete coverage of the human genome.Nat Genet. 2004; 36: 299-303Crossref PubMed Scopus (525) Google Scholar Before hybridization, 0.6 μg of fluorometry-quantified patient DNA and normal same-sex reference DNA were separately labeled with either Cy3 or Cy5 by random priming with separate fluor-conjugated 2-deoxycytidine 5-triphosphate and were purified from unincorporated nucleotides by spinning through a G-50 Sephadex column. Equal amounts of Cy3-labeled patient and Cy5-labeled reference DNA were mixed and precipitated with Cot 1 DNA, then resuspended in hybridization solution and applied to the array for 48 h. Arrays were washed in a 50% formamide solution and then were mounted in a 4',6-diamidino-2-phenylindole (DAPI) solution before imaging. Sixteen-bit 2,048×2,048 pixel DAPI, Cy3, and Cy5 images were collected using a custom CCD camera system,5Hamilton G Brown N Oseroff V Huey B Segraves R Sudar D Kumler J Albertson D Pinkel D A large field CCD system for quantitative imaging of microarrays.Nucleic Acids Res. 2006; 34: e58Crossref PubMed Scopus (19) Google Scholar and the data were analyzed, using UCSF SPOT15Jain AN Tokuyasu TA Snijders AM Segraves R Albertson DG Pinkel D Fully automatic quantification of microarray image data.Genome Res. 2002; 12: 325-332Crossref PubMed Scopus (291) Google Scholar to automatically segment the array spots and to calculate the log2 ratios of the total integrated Cy3 and Cy5 intensities for each spot. A second custom program, SPROC, applied quality criteria to the measurements and mapped them to the genome, currently with use of the May 2004 freeze of the draft sequence (UCSC Genome Bioinformatics hg17). The detailed protocols for each of these methods are available on the UCSF Comprehensive Cancer Center Web site. All aCGH results were confirmed using FISH or loss-of-heterozygosity (LOH) studies. For LOH analysis, DNA from the probands (subjects LR05-202, LR06-076, and LR02-409), mothers (LR05-202-1 and LR06-076-1), and fathers (LR05-202-2 and LR06-076-2) was amplified with primers for markers spanning from 215.36 Mb to 246.19 Mb within 1q4. Primer sequences are available at the UCSC Genome Bioinformatics Web site. Products were resolved and sized by capillary electrophoresis with use of an ABI 3730XL (Applied Biosystems). Deletion and breakpoint mapping by FISH was undertaken for patients LP94-079, LR04-249, LR02-409, and LR05-101. Chromosome preparations were produced from peripheral-blood lymphocytes by conventional techniques. BAC and fosmid clones from the regions of interest were identified from the Ensembl genome database. They were labeled with Spectrum Green or Spectrum Orange (Vysis), with use of nick translation. FISH was performed using standard methods, and images were captured using a cooled CCD camera and Smart Capture software (Applied Imaging). To provide confirmation for patients LP94-079, LR02-409, and LR05-101, each FISH experiment was undertaken using breakpoint-spanning clones for which 15 good-quality metaphase images were studied, as well as 5 or more good-quality metaphase images for the remaining clones. We had only a small archival sample for deceased patient LR04-249, which prevented the use of metaphase spreads. FISH signals were instead sought in interphase cells, and 5–10 cells were imaged. Primer sequences for the coding exons of AKT3 and ZNF238, as well as the ultraconserved element UC.43 located within intron 1 of AKT3,16Bejerano G Pheasant M Makunin I Stephen S Kent WJ Mattick JS Haussler D Ultraconserved elements in the human genome.Science. 2004; 304: 1321-1325Crossref PubMed Scopus (1158) Google Scholar were obtained from the Primer3 database (primers and PCR conditions are available on request). Amplicons were engineered to span the entire transcribed sequence of the two genes, including the 5′ and 3′ UTRs, and the entire reported sequence of UC.43. However, primers designed to sequence the final 249 bp of the coding sequence of ZNF238 did not amplify on genomic DNA, so this region was not sequenced. The products were purified using Sephacryl media (GE Healthcare) with Durapore (Millipore) polyvinylidene difluoride columns, and amplicons were then sequenced by the DNA Sequencing and Genotyping Core at the University of Chicago. The reactions used a 1/4 volume BigDye 3.1 terminator kit protocol, with standard cycling conditions, for 35 cycles with use of 1.2 μl template and 1.2 μl primer. Runs were done on the 3730XL instrument from Applied Biosystems, with use of standard 2-h runs. Sequence data were analyzed using Sequencher (Gene Codes) sequence-analysis software and were compared with annotated sequence from the National Center for Bioinformatics. Whole-mount in situ expression analysis was conducted on CD1 mouse embryos at embryonic day 10.5 (E10.5) and E12.5, as described elsewhere.17Chizhikov VV Millen KJ Control of roof plate formation by Lmx1a in the developing spinal cord.Development. 2004; 131: 2693-2705Crossref PubMed Scopus (71) Google Scholar For timed pregnancies, noon of the vaginal-plug date was E0.5. Antisense and sense probes were generated by in vitro transcription from Research Genetics mouse IMAGE clones 6417039 (Znf238) and 30089997 (Akt3), which were first sequenced to confirm identity. We studied a group of six patients with deletions of distal 1q42-q44 that are all associated with developmental brain malformations (table 1 and figs. 1 and 2).Table 1OFC and Brain Malformations in Patients with 1q4 DeletionOFC SDbOr percentile.PatientaPatients other than those from the present study are listed by the authors who reported them.AKT3At BirthLater (Age)Brain MalformationsGentile et al.18Gentile M Di Carlo A Volpe P Pansini A Nanna P Valenzano MC Buonadonna AL FISH and cytogenetic characterization of a terminal chromosome 1q deletion: clinical case report and phenotypic implications.Am J Med Genet A. 2003; 117: 251-254Crossref Scopus (39) Google ScholarDeleted−2−4 (8 mo)MIC and partial ACCVan Bever et al.19van Bever Y Rooms L Laridon A Reyniers E van Luijk R Scheers S Wauters J Kooy RF Clinical report of a pure subtelomeric 1qter deletion in a boy with mental retardation and multiple anomalies adds further evidence for a specific phenotype.Am J Med Genet A. 2005; 135: 91-95Crossref PubMed Scopus (51) Google ScholarDeleted−3NAMIC, partial ACC, and CVHLR04-249Deleted−4NAMIC, ACC, and CVHLR05-202Deleted−3−5 (2 years)MIC, ACC, and CVHLR02-409Deleted−5cAt age 15 wk.−7 (5 years)MIC, partial ACC, and CVHLR06-076Deleted−2dAt age 4 mo.−3.5 (4 years)Mild MIC and partial ACCLR05-101Disrupted50th−2.5 (2 years)MIC and ACCde Vries et al.20de Vries BB Knight SJ Homfray T Smithson SF Flint J Winter RM Submicroscopic subtelomeric 1qter deletions: a recognisable phenotype?.J Med Genet. 2001; 38: 175-178Crossref PubMed Google ScholarNot testedNA−5 (2.5 years)MIC and partial ACCde Vries et al.21de Vries BB White SM Knight SJ Regan R Homfray T Young ID Super M McKeown C Splitt M Quarrell OW et al.Clinical studies on submicroscopic subtelomeric rearrangements: a checklist.J Med Genet. 2001; 38: 145-150Crossref PubMed Scopus (245) Google ScholarNot tested−2−5 (1 year)MIC and partial ACCLP94-079Not deleted10thNACVH and PMGDaniel et al.22Daniel A Baker E Chia N Haan E Malafiej P Hinton L Clarke N Adès L Darmanian A Callen D Recombinants of intrachromosomal transposition of subtelomeres in chromosomes 1 and 2: a cause of minute terminal chromosomal imbalances.Am J Med Genet A. 2003; 117: 57-64Crossref Scopus (19) Google ScholarNot deletedNormalNormal (15 years)None seen on CT scanNote.—NA=not available.a Patients other than those from the present study are listed by the authors who reported them.b Or percentile.c At age 15 wk.d At age 4 mo. Open table in a new tab Figure 2Brain-imaging abnormalities in patients with deletion 1q4, shown with head CT scan (A–C) or with T1-weighted midline sagittal (D) and T2-weighted axial (E and F) MRI sequences in patients LP94-079 (A–C) and LR04-249 (D–F). In patient LP94-079, the cortex appears thickened in the posterior frontal and perisylvian regions (white arrow[s] in panels A–C), suggesting PMG, although the resolution is low. The lateral ventricles are located in the normal position close to the midline (A and B), suggesting that the corpus callosum is normal. The cerebellar vermis is small, since it is not seen at the level of the low midbrain (white arrowhead in panel A), and a small skull defect is seen beneath an occipital cephalocele (white arrowhead in panel C). In LR04-249, gyral pattern is poorly developed, even for 35 wk gestation, and the corpus callosum is absent (white arrow in panel D and two black arrows in panel E). The cerebellar vermis is small, with the inferior margin (dashed line in panel D) well above the level of the obex (solid line in panel D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Note.— NA=not available. This girl was born at term, with a birth weight of 2.9 kg, length of 48 cm (both 25th percentile), and an occipitofrontal circumference (OFC) of 31.75 cm (10th percentile), and she had low blood glucose. Family history was significant for multiple miscarriages in close relatives and a paternal cousin who died with probable hydrocephalus; the pregnancy was complicated by polyhydramnios. Examination demonstrated a high forehead with vertical furrowing, hypertelorism, mildly large tongue, small jaw, low posterior hairline, widely spaced nipples, mild rhizomelic shortening of all limbs, short fingers with mildly broad distal phalanges, fifth-finger clinodactyly, abnormal palmar and plantar creases, and anteriorly displaced anus. She did not have organomegaly. Echocardiogram demonstrated patent foramen ovale, secundum atrial septal defect, small patent ductus, and moderate tricuspid valve insufficiency. Cranial CT scan showed thick and irregular cortex, consistent with PMG in the posterior frontal and perisylvian regions, best seen on the right, as well as severe CVH and a small occipital cephalocele (fig. 2A–2C). The corpus callosum appeared normal. She died at age 12 d from complications of supraventricular tachycardia. This boy was born prematurely, at 35 wk gestation, because of preterm labor. His birth weight was 1.35 kg (−3 SD), length was 36.5 cm (−4 SDs), and OFC was 25 cm (−4 SD). His primary medical problems were intestinal obstruction, anemia secondary to ABO incompatability, and increasingly frequent episodes of desaturation, which led to his death at age 6 wk. He had obvious growth retardation, including severe MIC, low-set ears, long smooth philtrum, thin lips, small jaw, high-narrow palate, severe hypospadias, undescended testes, and sacral dimple. Further tests demonstrated patent foramen ovale, mild right-atrial enlargement, abnormal “domed” pulmonary valve, dilatation of the right pulmonary artery, microcolon with intestinal obstruction, small kidneys with renal insufficiency, 13 pairs of ribs, and butterfly vertebrae at T5 and T10. Brain magnetic resonance imaging (MRI) showed a very immature gyral pattern, even for 35 wk gestation, that suggests a possible cortical malformation (type uncertain); mildly enlarged and widely separated lateral ventricles; total ACC; and moderate CVH (fig. 2D–2F). This girl was born at term after a pregnancy complicated by abnormal triple-screen results. However, level II ultrasound was normal, so the family did not proceed with amniocentesis. At birth, she had MIC with a birth OFC of 30.5 cm (−3 SD), two ventricular septal defects, and a single palmar crease. OFC was 38.5 cm at age 7 mo (−4 SD) and 41 cm at age 2 years (−5 SD). She was noted to have delays in cognitive, fine and gross motor, and social skills. Seizures began at age 8.5 mo and led to an evaluation that included brain MRI. At age 2.5 years, her seizures were well controlled with phenobarbitol treatment. A review of systems revealed corrected myopia; three small ventricular septal defects, two of which had closed; mild eczema; history of urinary-tract infections; mildly distended right renal pelvis on kidney ultrasound; and allergy to milk. She had hypotonia, a broad nasal bridge, low-set ears, short webbed neck, epicanthal folds, and small jaw. She was able to crawl, pull herself to a standing position, and vocalize, although she does not use intelligible words. Brain MRI at age 8 mo revealed a normal gyral pattern, total ACC, mildly enlarged third and lateral ventricles, markedly reduced white-matter volume, small bilateral Probst bundles, and mild CVH (fig. 1D–1F). This boy was born at term; birth OFC is not available, although it was described as borderline small. His OFC was 39.5 cm at age 4 mo (−2 SD), 41 cm at age 9 mo, 45 cm at age 30 mo, and 46 cm at age 4 years (last three values approximately −3.5 SD). Generalized seizures began at age 2.5 years and have been partially controlled with phenobarbital and valproic acid treatment. On examination at age 4 years, he had bilateral ear pits, small jaw, bilateral supernumerary nipples, abnormal foreskin of the penis, undescended left testicle, intermittent esotropia, and generalized spasticity with brisk reflexes. He did not understand speech and was not able to walk or talk. Brain MRI demonstrated a normal gyral pattern, diffusely decreased white-matter volume, partial ACC with thin posterior body and absent splenium, and normal cerebellum (fig. 1G–1I). This girl was born at 36 wk gestation by emergency cesarean section because of placenta previa, with a birth weight 1.9 kg and length of 42 cm (both 3rd percentile). Her OFC was 33.3 cm at age 15 wk (−4 SD for corrected age of 11 wk) and fell to −5 SD by age 1 year. She began having seizures at age 8 mo. At age 5 years, she was small for her age, with weight of 10.45 kg (−4 SD), length of 93 cm (−3 SD), and OFC of 41.5 cm (−7 SD). She was unable to walk or use any words, suggesting severe mental retardation. On examination, she was strikingly dysmorphic, with MIC, bitemporal narrowing, full cheeks, prominent epicanthal folds, anteverted nares, and tented upper lip with a long philtrum. She also had a mixed sensorineural and conductive hearing loss, left esotropia, and atrial septal defect. Brain MRI demonstrated a normal gyral pattern, partial ACC with thin body and absent splenium, and mild CVH (fig. 1J–1L). This boy was born at term after an uncomplicated pregnancy, except that a prenatal ultrasound at 22 wk gestation did not detect the septum pellucidum. His birth weight was 3.88 kg (75th percentile), length was 54 cm (90th percentile), and OFC was 35 cm (50th percentile). Developmental delay and generalized hypotonia were noted at age 3 mo because of lack of head control and visual tracking, and seizures began at age ∼1 year. By age 2 years, he had global developmental delay, axial hypotonia, limb spasticity, and poor feeding that required placement of a nasogastric tube. He was not dysmorphic. Examination revealed a small penis. Serial exams demonstrated postnatal MIC, and his OFC was at the 25th percentile by age 3 mo and at the 2nd percentile by age 12 mo. His OFC at age 28 mo was 46.3 cm (−2.5 SD). Brain MRI demonstrated a normal gyral pattern, reduced white matter, mildly enlarged and widely separated lateral ventricles, complete ACC with Probst bundles along the medial hemispheric walls, and mild CVH (fig. 1M–1O). We mapped the 1q4 breakpoints of all six patients by either BAC and fosmid FISH or by aCGH (fig. 3). Chromosome analysis and FISH detected an unbalanced translocation inherited from the proband's father: 46,XX,der(1)t(1;11)(q44;p15.3)pat. The extent of the deletion was determined by mapping the chromosome 1 breakpoint of the balanced translocation in the father. The clone RP11-518I10 (242.57–242.65 Mb) at 1q44 was split between the translocation derivatives, indicating that all DNA telomeric to this probe was present in one copy in this child (fig. 3). Chromosome analysis showed a large interstitial deletion: 46,XY,del(1)(q42.1q44) de novo. FISH analysis demonstrated that the deletion extended from BAC clone RP11-87P4 (230.78–230.9 Mb) to RP11-399B15 (243.22–243.42 Mb), a region of >13 Mb. Chromosome analysis was interpreted as normal, but telomeric probes detected a de novo deletion of 1qter. aCGH demonstrated a large subtelomeric de novo deletion beginning after BAC RP11-147E13 (ending at 235.38 Mb) and extending to the telomere, a distance of 11.9 Mb (fig. 4). This deletion was also confirmed using LOH analysis with STRP (microsatellite) markers beginning with marker D1S227 (215.36 Mb) and extending to the subtelomeric marker D1S2682 (246.19 Mb). Some of these interspersed markers were uninformative, since both parents carried the same allele(s), but, for all of these, the patient was homozygous for an allele carried by the mother, consistent with the pattern established by the unambiguously deleted markers (table 2). Note.— Allele sizes from the probands (LR05-202, LR06-076, and LR02-409), mothers (LR05-202-1 and LR06-076-1), and fathers (LR05-202-2 and LR06-076-2) are listed for each marker (with associated interpretation). No DNA was available at the time of the study for the parents of proband LR02-409, so only heterozygosity is noted. Chromosome numbering is derived from the hg18 genome assembly (March 2006). Maternal=maternally inherited region. Chromosome analysis was normal, but aCGH detected a small deletion of 1q44 between BACs RP11-158M3 (237.51–237.68 Mb) and RP11-606D18 (245.33–245.50 Mb), demonstrating a deletion of 7.6A Mb between 237.68 Mb and 245.33 Mb. This interstitial deletion was confirmed by a second aCGH analysis and by LOH analysis (fig. 4 and table 2). The initial chromosome analysis detected an apparently balanced paracentric inversion of distal 1q. In view of the girl's phenotype, chromosome analysis was repeated and demonstrated a complex rearrangement of distal 1q, with two paracentric inversions 17.36 Mb apart—inv(1)(q32.2q42.2) and inv(1)(q43q44)—each containing a small interstitial deletion. The proximal inversion extends from RP11-434B7 (211.48–211.60 Mb) in 1q32.2 to RP11-99J16 (228.96–229.01 Mb) in 1q42.2, whereas the distal inversion extends from RP11-433N10 (236.57–236.76 Mb) in 1q43 to CTB-160H23 at 1q44. In addition, we found two deletions in the region. The proximal 4-Mb deletion was located in 1q41-q42.12 and included the clones RP11-332J14 (218.70–218.87 Mb) and RP11-192M1 (224.12–224.28 Mb). The distal deletion was located in 1q43-q44. It included the clones RP11-553N16 (240.00–240.15 Mb) and RP1-241M7 (242.49–242.57 Mb) and was flanked by the nondeleted clones RP11-80B9 (238.96–239.13 Mb) and RP11-518L10 (242.57–242.65 Mb). The deletion is therefore 200 bp with 100% conservation among human, rat, and mouse.16Bejerano G Pheasant M Makunin I Stephen S Kent WJ Mattick JS Haussler D Ultraconserved elements in the human genome.Science. 2004; 304: 1321-1325Crossref PubMed Scopus (1158) Google Scholar All regions were sequenced in at least 47 patients. No pathogenic mutations were defined. The sequence of UC.43 was normal in 43 patients. The remaining 4 of 47 patients, as well as 1 of 8 control subjects, had a heterozygous SNP (C→T at position 111153 of AL662889). In addition, we sequenced the first 1,315 of 1,566 bp of the coding region of the single exon of ZNF238 and found a heterozygous synonymous substitution (val74val GTG→GTA) in one patient. Sequencing of AKT3 and ZNF238 for patient LR05-101 with the t(1;13) translocation did not define any sequence changes in either the coding region or UTRs of either gene. We were therefore unable to test for monoallelic versus biallelic expression of either gene in this patient. RT-PCR and northern analysis data indicate that Akt3
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