Background Great Ormond Street Hospital cares for many children with rare and complex disease. A substantial proportion of these children will have an underlying genetic cause for their condition. Making a diagnosis in such cases is increasingly achievable due to advances in genomic testing. However, such testing may not be available on the NHS and results, when accessed in the research setting, typically take many months to report. In critically ill children, cared for on the intensive care units, a timely genetic diagnosis may be beneficial on a number of levels. It may inform treatment options and prognosis. It may also reduce the need for further investigations, improving the diagnostic odyssey and potentially reducing costs. In addition, it may help the family understand recurrence risks. Methods To address this unmet need we developed a work flow to enable Rapid Paediatric Sequencing (RaPS) in critically ill children. Eligible patient-parent trios were recruited and underwent whole genome sequencing. A framework was developed to allow capture of clinical information and robust analysis of genomic data initially by investigating candidate gene lists and HPO-derived panels. If no genetic diagnosis was forthcoming, genomic analysis was broadened and more disease-causing genes were investigated. Results Twenty two patient-parent trios were recruited to the RaPS pilot project. A diagnosis was reached in 8 cases (36%). The shortest turnaround time from sample collection to return of provisional results was 5 days. A genetic diagnosis informed point of care decisions in at least 3 cases. Conclusion We report on the process and framework necessary to deliver rapid whole genome sequencing to critically ill paediatric patients and reflect on the outcomes, clinical utility and challenges of implementing such a service at GOSH.

Background: Osteogenesis imperfecta (OD, the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit.Report: Whole exome sequencing in patients with 01 identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>Tp.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(G1n678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency.Discussion: Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from 'Classical' OI.Conclusions: Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients. (C) 2016 Elsevier Inc. All rights reserved.

aOxford Centre for Genomic Medicine, Nuffield Orthopaedic Centre, Oxford University Hospitals NHS Foundation Trust, Headington bDepartment of Clinical Genetics, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK Correspondence to Henrietta Lefroy, BSc, MBChB, MRCP, Oxford Centre for Genomic Medicine, Nuffield Orthopaedic Centre, Oxford University Hospitals NHS Foundation Trust, ACE Building, Windmill Road, Headington OX3 7HE, Oxford, UK Tel: +44 186 522 6024; e-mail: [email protected]

We identified, by homozygosity mapping, a novel locus on 10q21.3- q22.1 for Goldberg- Shprintzen syndrome ( GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development.

We identified, by homozygosity mapping, a novel locus on 10q21.3-q22.1 for Goldberg-Shprintzen syndrome (GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development. We identified, by homozygosity mapping, a novel locus on 10q21.3-q22.1 for Goldberg-Shprintzen syndrome (GOSHS) in a consanguineous Moroccan family. Phenotypic features of GOSHS in this inbred family included microcephaly and mental retardation, which are both central nervous system defects, as well as Hirschsprung disease, an enteric nervous system defect. Furthermore, since bilateral generalized polymicogyria was diagnosed in all patients in this family, this feature might also be considered a key feature of the syndrome. We demonstrate that homozygous nonsense mutations in KIAA1279 at 10q22.1, encoding a protein with two tetratrico peptide repeats, underlie this syndromic form of Hirschsprung disease and generalized polymicrogyria, establishing the importance of KIAA1279 in both enteric and central nervous system development. Goldberg-Shprintzen syndrome (GOSHS) (Goldberg and Shprintzen Goldberg and Shprintzen, 1981Goldberg RB Shprintzen RJ Hirschsprung megacolon and cleft palate in two sibs.J Craniofac Genet Dev Biol. 1981; 1: 185-189PubMed Google Scholar) is a disorder characterized by microcephaly, mental retardation, facial dysmorphism, and Hirschsprung disease (HSCR). GOSHS shares the same accession number (MIM 235730) in the Online Mendelian Inheritance in Man (OMIM) database as the comparable disorder Mowat-Wilson syndrome (MWS) (Mowat et al. Mowat et al., 1998Mowat DR Croaker GD Cass DT Kerr BA Chaitow J Ades LC Chia NL Wilson MJ Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22-q23.J Med Genet. 1998; 35: 617-623Crossref PubMed Scopus (197) Google Scholar). Genetically, however, they are different. MWS occurs as a de novo dominant syndrome, whereas GOSHS is most likely inherited as an autosomal recessive trait, a hypothesis based on the occurrence of the disease in consanguineous pedigrees and in affected siblings with unaffected parents (Hurst et al. Hurst et al., 1988Hurst JA Markiewicz M Kumar D Brett EM Unknown syndrome: Hirschsprung's disease, microcephaly, and iris coloboma: a new syndrome of defective neuronal migration.J Med Genet. 1988; 25: 494-497Crossref PubMed Scopus (55) Google Scholar; Brooks et al. Brooks et al., 1999Brooks AS Breuning MH Osinga J vd Smagt JJ Catsman CE Buys CH Meijers C Hofstra RM A consanguineous family with Hirschsprung disease, microcephaly, and mental retardation (Goldberg-Shprintzen syndrome).J Med Genet. 1999; 36: 485-489PubMed Google Scholar). MWS is associated with de novo mutations in ZFHX1B, located at 2q22 (Amiel et al. Amiel et al., 2001Amiel J Espinosa-Parrilla Y Steffann J Gosset P Pelet A Prieur M Boute O Choiset A Lacombe D Philip N Le Merrer M Tanaka H Till M Touraine R Toutain A Vekemans M Munnich A Lyonnet S Large-scale deletions and SMADIP1 truncating mutations in syndromic Hirschsprung disease with involvement of midline structures.Am J Hum Genet. 2001; 69: 1370-1377Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar; Wakamatsu et al. Wakamatsu et al., 2001Wakamatsu N Yamada Y Yamada K Ono T Nomura N Taniguchi H Kitoh H Mutoh N Yamanaka T Mushiake K Kato K Sonta S Nagaya M Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease.Nat Genet. 2001; 27: 369-370Crossref PubMed Scopus (262) Google Scholar; Zweier et al. Zweier et al., 2002Zweier C Albrecht B Mitulla B Behrens R Beese M Gillessen-Kaesbach G Rott HD Rauch A "Mowat-Wilson" syndrome with and without Hirschsprung disease is a distinct, recognizable multiple congenital anomalies-mental retardation syndrome caused by mutations in the zinc finger homeo box 1B gene.Am J Med Genet. 2002; 108: 177-181Crossref PubMed Scopus (105) Google Scholar), whereas, for GOSHS, no causative gene has yet been identified. Also, some clinical differences exist. In patients with MWS, neurological abnormalities such as epilepsy (in a high percentage of patients), agenesis of the corpus callosum (in 35% of patients), and cortical malformations (in a minority of patients) have been reported (Amiel et al. Amiel et al., 2001Amiel J Espinosa-Parrilla Y Steffann J Gosset P Pelet A Prieur M Boute O Choiset A Lacombe D Philip N Le Merrer M Tanaka H Till M Touraine R Toutain A Vekemans M Munnich A Lyonnet S Large-scale deletions and SMADIP1 truncating mutations in syndromic Hirschsprung disease with involvement of midline structures.Am J Hum Genet. 2001; 69: 1370-1377Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar; Zweier et al. Zweier et al., 2002Zweier C Albrecht B Mitulla B Behrens R Beese M Gillessen-Kaesbach G Rott HD Rauch A "Mowat-Wilson" syndrome with and without Hirschsprung disease is a distinct, recognizable multiple congenital anomalies-mental retardation syndrome caused by mutations in the zinc finger homeo box 1B gene.Am J Med Genet. 2002; 108: 177-181Crossref PubMed Scopus (105) Google Scholar; Silengo et al. Silengo et al., 2004Silengo M Ferrero GB Wakamatsu N Pachygyria and cerebellar hypoplasia in a patient with a 2q22-q23 deletion that includes the ZFHX1B gene.Am J Med Genet A. 2004; 127: 109Crossref Google Scholar). In the few patients with GOSHS discussed in published reports, neurological symptoms have not been studied in detail, and the cause of the microcephaly and mental retardation remains obscure (Goldberg and Shprintzen Goldberg and Shprintzen, 1981Goldberg RB Shprintzen RJ Hirschsprung megacolon and cleft palate in two sibs.J Craniofac Genet Dev Biol. 1981; 1: 185-189PubMed Google Scholar; Hurst et al. Hurst et al., 1988Hurst JA Markiewicz M Kumar D Brett EM Unknown syndrome: Hirschsprung's disease, microcephaly, and iris coloboma: a new syndrome of defective neuronal migration.J Med Genet. 1988; 25: 494-497Crossref PubMed Scopus (55) Google Scholar). Here, we describe a family with HSCR (an anomaly of the enteric nervous system that is of neural crest origin and is histologically characterized by the absence of ganglion cells in the myenteric and submucosal plexuses) (Okamoto and Ueda Okamoto and Ueda, 1967Okamoto E Ueda T Embryogenesis of intramural ganglia of the gut and its relation to Hirschsprung's disease.J Pediatr Surg. 1967; 2: 437-443Abstract Full Text PDF Scopus (239) Google Scholar) as a variable feature and bilateral generalized polymicrogyria (PMG) (a developmental malformation of the cerebral cortex, characterized by an enlarged number of smaller convolutions or gyri and disruption of the normal six-layered cerebral cortical structure) (Friede Friede, 1989Friede RL Developmental neuropathology. 2nd ed. Springer Verlag, Berlin1989Crossref Google Scholar) as a constant feature. The established diagnosis for this family was GOSHS, although bilateral generalized PMG (BGP) has not been reported as part of the syndrome before. To unravel the genetic basis of GOSHS, we performed a complete genome scan and homozygosity mapping in a large consanguineous Moroccan family reported elsewhere (Brooks et al. Brooks et al., 1999Brooks AS Breuning MH Osinga J vd Smagt JJ Catsman CE Buys CH Meijers C Hofstra RM A consanguineous family with Hirschsprung disease, microcephaly, and mental retardation (Goldberg-Shprintzen syndrome).J Med Genet. 1999; 36: 485-489PubMed Google Scholar). The segregation of the disease in the pedigree is consistent with an autosomal recessive mode of inheritance, with several consanguineous loops (fig. 1), and the family therefore was considered suitable for homozygosity mapping (Lander and Botstein Lander and Botstein, 1987Lander E Botstein D Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children.Science. 1987; 236: 1567-1570Crossref PubMed Scopus (686) Google Scholar). Informed consent was obtained from the parents of all subjects. In this family, five patients had the cardinal signs characterizing GOSHS (one boy lacked HSCR). One child with long-segment HSCR died in the neonatal period; his DNA was unavailable for our study. When the youngest patient was diagnosed, brain magnetic resonance imaging (MRI) revealed BGP. MRI scans of all living patients also revealed BGP (fig. 2). Gait disturbance, speech defect, excessive drooling, and pseudobulbar signs indicate diffuse cortical dysfunction including the perisylvian areas.Figure 2Representative MRIs of patient VI-1 (panels A and B) and patient VI-10 (panels C and D), showing BGP. A, Axial T2-weighted MRI of patient VI-1 at age 5 mo, showing frontoparietal and occipital bilateral PMG. B, Coronal FLAIR image of the same patient showing perisylvian PMG (arrow). C, T1-weighted axial image of patient VI-10 at age 14 years, showing BGP with distribution similar to that seen in patient VI-1. D, Parasagittal T1-weighted image of patient VI-10, showing PMG of the right peri-insular temporal lobe.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Genomic DNA was isolated from peripheral leukocytes by use of the method described by Miller et al. (Miller et al., 1988Miller SA Dykes DD Polesky HF A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17845) Google Scholar). By haplotyping, FISH, and sequence analyses, we excluded the ZFHX1B gene as a cause of GOSHS in this family (data not shown). For the systematic genome scan, 381 markers (STRPs) from the ABI Prism Linkage Mapping Set MD-10 (version 2.5), with an average spacing of 10 cM, were tested. DNA amplification was performed using 25 ng genomic DNA in 7.5−μl PCRs containing 1× PCR Gold Buffer, 2.5 mM MgCl2, 10 μM primer pair mix, and 0.4 U AmpliTaq Gold DNA polymerase (Applied Biosystems). Amplification conditions were 10 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min 30 s at 72°C; amplification was ended by a final extension for 5 min at 72°C. PCR products were pooled in panels and were loaded on an ABI 3100 automated sequencer. Data were analyzed using GeneMapper software (version 2.1) (Applied Biosystems). After this initial screening, a region on 10q21.3-q22.1 was further investigated, since one of the four affected individuals was homozygous by descent for two consecutive markers, D10S1652 and D10S537, whereas the other three patients were homozygous for one of these two markers. No other regions of homozygosity for consecutive markers were identified. We saturated the region between markers D10S1652 and D10S537 (12.3 cM) with 11 additional markers; they either were obtained from the Marshfield genetic map (Center for Medical Genetics Web site) or were newly developed. Marker order and genetic distances were obtained from the Marshfield genetic map (Center for Medical Genetics Web site) and the Celera physical map. All patients were homozygous for 7 of these 11 markers. A recombination event between loci CGR166 and CGR167 in patient VI-7 defined the centromeric boundary of the genetic interval. The telomeric border was defined by recombination events between markers D10S1665 and CGR170 in patients VI-1 and VI-3 (fig. 1). These recombination events reduced the candidate region to a minimum of 2.8 Mb and a maximum of 3.8 Mb. Parametric two-point and multipoint linkage analyses were performed using the MLINK program from the LINKAGE (version 5.1) software package (Lathrop and Lalouel Lathrop and Lalouel, 1984Lathrop GM Lalouel JM Easy calculations of LOD scores and genetic risks on small computers.Am J Hum Genet. 1984; 36: 460-465PubMed Google Scholar) and the SimWalk2 program (version 2.9) (Sobel et al. Sobel et al., 2002Sobel E Papp JC Lange K Detection and integration of genotyping errors in statistical genetics.Am J Hum Genet. 2002; 70: 496-508Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). LOD scores were calculated, under the assumption that the disease phenotype is a rare autosomal recessive disorder with 100% penetrance, with a gene frequency of 1:5,000. No phenocopies were allowed, and equal allele frequencies were used because of the limited number of available independent family members. A significant two-point LOD score was obtained (max LOD score 3.29 and θ=0 for marker CGR167) (table 1), which increased to a maximum multipoint LOD score of 5.9 between markers CGR167 and D10S1647 (fig. 3).Table 1Results of Two-Point LOD Score Analysis, Performed for Markers Used in Fine MappingLOD at θ =MarkeraMarker order follows that of the Celera physical map.0.01.05.1.2.3.4D10S196−13.81−3.96−1.52−.65−.08.020D10S1652−6.96−2.39−1.85−1.36−.68−.35−.15D10S1743−4.9711.391.31.9.46.14CGR166−3.7.581.031.02.73.4.13CGR1673.29bMax LOD score.3.212.92.511.731.4D10S2102.592.532.261.921.26.67.24D10S16781.491.451.291.09.72.4.16D10S16471.841.791.581.33.86.47.19D10S16722.882.812.522.151.42.76.25D10S16652.772.72.432.081.39.75.25CGR1721.361.321.17.97.62.32.11CGR170−.281.591.971.881.4.83.33D10S676−.281.251.641.561.1.59.18D10S537−.111.451.821.711.2.65.21a Marker order follows that of the Celera physical map.b Max LOD score. Open table in a new tab The maximum 3.8-Mb linked region of shared homozygosity (defined by CGR166 and CGR170) contained a total of 25 known genes and 5 genes encoding hypothetical proteins, in accordance with the National Center for Biotechnology Information (NCBI) build 34 of the human genome and the Ensembl Genome Browser (table 2). We first sequenced seven functional candidate genes on the basis of their putative involvement in neurogenesis (CTNNA3, LRRTM3, ATOH7, DDX50, DDX21, NET-7, and NEUROG3), either using cDNA obtained by RT-PCR from a lymphoblastoid cell line of patient VI-10 and from a control or using genomic DNA from both. Mutations were not identified. We then performed a systematic mutational screening of all transcripts from the candidate region. PCR products from either genomic DNA (exons including flanking intronic sequences) or reverse-transcribed products (ORFs) were amplified and purified (ExoSapIT [USB]). PCR primers, which were designed using standard software (Primer3), and conditions are available on request. Bidirectional sequencing was performed using BigDye Terminator chemistry on an ABI 3100 sequencer (Applied Biosystems). With the use of the software package SeqScape (version 2.1) (Applied Biosystems), sequences were aligned and compared with consensus sequences.Table 2Gene and Marker Positions and DetailsGene or MarkerCelera Position (Mb)Additional DetailsD10S196…GenBank accession number Z16598D10S1652…GenBank accession number Z52339D10S174360.5GenBank accession number Z53951 (located at BAC AC020641)CTNNA360.95–62.735′→3′: catenin (cadherin-associated protein), alpha 3CGR16661.2GenBank accession number BV212297 (located at BAC AC27675)LRRTM361.9–62.13′→5′: leucine-rich repeat transmembrane neuronal 3CGR16761.63–62.1GenBank accession number BV212296 (located at BAC AC084303)LOC340888…3′→5′: similar to aldo-keto reductase family 1, member B10; aldose reductase-like 1 pseuodogeneJDP162.83–62.875′→3′: J domain–containing protein 1LOC389974…3′→5′: similar to 60S ribosomal protein L12SIRT162.91–62.953′→5′: sirtuin (silent mating type information regulation 2 homolog) 1 (S. cerevisiae)DKFZP564G092…5′→3′: DKFZP564G092 protein, HERC4MYPN63.14–63.243′→5′: myopalladinATOH763.26–63.265′→3′: atonal homolog 7 (Drosophila)LOC389975…3′→5′: similar to keratin 19; keratin, type I cytoskeletal 19; keratin, type I, 40-kd; pseudogeneMAWBP63.31–63.365′→3′: MAWD-binding proteinD10S21063.32GenBank accession number Z16813 (located at BAC AC016395)HNRPH3…3′→5′: heterogeneous nuclear ribonucleoprotein H3 (2H9)RUFY263.37–63.445′→3′: RUN and FYVE domain–containing 2DNA2L63.44–63.505′→3′: DNA2 DNA replication helicase 2-like (S. cerevisiae)SLC25A1663.51–63.565′→3′: solute carrier family 25CXXC663.68–63.723′→5′: CXXC finger 6CCAR163.75–63.83′→5′: cell division cycle and apoptosis regulator 1C10orf2463.92–63.933′→5′: chromosome 10 ORF 24: ORF with an as-yet-unknown functionD10S167863.8–64GenBank accession number Z52660 (located at BAC AL359844)DDX5063.93–63.983′→5′: DEAD (Asp-Glu-Ala-Asp) box polypeptide 50DDX2163.99–63.023′→5′: DEAD (Asp-Glu-Ala-Asp) box polypeptide 21KIAA127964.02–64.053′→5′: KIAA1279LOC389976…3′→5′: similar to actin 3 (Drosophila) (fragments) pseudogenePRG164.12–64.143′→5′: proteoglycan 1, secretory granuleVPS2664.1–64.23′→5′: vacuolar protein–sorting 26 (S. cerevisiae)SUPV3L164.22–64.243′→5′: suppressor of var1, 3-like 1 (S. cerevisiae)FLJ2276164.2–64.33′→5′: hypothetical protein FLJ22761D10S164764.20–64.38GenBank accession number Z52188 (located at BAC AL596223)HK164.3–64.433′→5′: hexokinase 1TACR264.44–64.455′→3′: tachykinin receptor 2NET-764.48–64.543′→5′: transmembrane 4 superfamily member tetraspan NET-7NEUROG364.6–64.65′→3′: neurogenin 3C10orf35…3′→5′: chromosome 10 ORF 35LOC389977…3′→5′: similar to ribosomal protein L5; 60S ribosomal protein L5 pseudogeneCOL13A164.8–64.93′→5′: collagen, type XIII, alpha 1LOC387686…5′→3′: LOC399779CGR17065GenBank accession number BV212295 (located at BAC AC024601)H2AFY265.0–65.13′→5′: H2A histone family, member Y2AMID65.15–65.185′→3′: apoptosis-inducing factor–homologous mitochondrion-associated inducer of deathD10S537…5′→3′ Open table in a new tab Analysis of 98% of all coding sequences from the region revealed one likely disease-causing homozygous nucleotide substitution, 303C→T, in exon 1 of the KIAA1279 gene (fig. 4A). This transversion leads, at the amino acid level, to the replacement of an arginine with a stop codon (R90X), resulting in a shortened protein of 89 aa. The 303C→T mutation showed complete segregation with the disease in the family. One hundred ethnically matched control chromosomes (of North African origin) were screened for the R90X mutation, and none of the controls carried this mutation. Subsequently, one additional family of British Pakistani ancestry, with a phenotype similar to that found in the Moroccan family, was screened for mutations in this gene. This family had four affected individuals with multiple inbreeding loops. Clinical characteristics were consistent with GOSHS, and CT brain scans of this family were described in a previous study (Hurst et al. Hurst et al., 1988Hurst JA Markiewicz M Kumar D Brett EM Unknown syndrome: Hirschsprung's disease, microcephaly, and iris coloboma: a new syndrome of defective neuronal migration.J Med Genet. 1988; 25: 494-497Crossref PubMed Scopus (55) Google Scholar) and were thought to represent an abnormality of neuronal migration. In the affected individuals, a homozygous G→T transversion was identified at nt 285, resulting, at the amino acid level, in the replacement of a glutamic acid with a stop codon (E84X) (fig. 4B). Two different homozygous nonsense mutations in KIAA1279 in two independent families with GOSHS strongly indicate that KIAA1279 is the gene responsible for GOSHS. KIAA1279 consists of seven exons spanning 28 kb of genomic DNA. The transcript encodes a protein of 621 aa. We identified orthologs of human KIAA1279 in fruit fly, frog, rat, mouse, bee, chicken, and Japanese puffer fish, using BLAST comparison (NCBI Web site). There is strong sequence conservation during evolution; the protein sequence of human KIAA1279 shares 89% amino acid identity with mouse product, 88% with rat product, and 29% with fruit fly product. Multitissue northern blots (both adult and fetal) (fig. 5) showed ubiquitous expression of the 2.4-kb KIAA1279 mRNA without a specific pattern, with expression lacking only in blood leukocytes (fig. 5A). In addition, northern blot hybridization identified KIAA1279 mRNA molecules in different parts of the adult CNS—namely, cerebellum, cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, and putamen (fig. 5B). This widespread KIAA1279 expression is consistent with data available in gene expression databases such as the Gene Expression Omnibus, the Human Unidentified Gene-Encoded (HUGE) Protein Database, and the University of California–Santa Cruz (UCSC) Genome Browser. The function of KIAA1279 is unknown. Neither the gene nor its derived protein shows any significant sequence similarity to known human cDNA or protein sequences. Possible aspects of the molecular function of KIAA1279 may be gleaned from its primary sequence. We predicted protein motifs and domains using the program SMART (Simple Modular Architecture Research Tool). The predicted protein contains two tetratrico peptide repeats (TPRs), which are structural motifs consisting of 34 amino acid residues, in exon 1/2 and exon 4, respectively. Because of these repeats, KIAA1279 may be considered a member of the TPR protein family (D'Andrea and Regan D'Andrea and Regan, 2003D'Andrea LD Regan L TPR proteins: the versatile helix.Trends Biochem Sci. 2003; 28: 655-662Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar). The most basic function of TPR motifs is to mediate protein-protein interactions. Proteins with TPR motifs are involved in a variety of biological processes, such as cell-cycle regulation, transcriptional control, mitochondrial and peroxisomal protein transport, neurogenesis, and protein folding. Several diseases have been described as being caused by mutations in genes encoding proteins containing TPR domains—for example, Leber congenital amaurosis (AIPL1) (Sohocki et al. Sohocki et al., 2000Sohocki MM Bowne SJ Sullivan LS Blackshaw S Cepko CL Payne AM Bhattacharya SS Khaliq S Qasim Mehdi S Birch DG Harrison WR Elder FF Heckenlively JR Daiger SP Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis.Nat Genet. 2000; 24: 79-83Crossref PubMed Scopus (234) Google Scholar) and Charcot-Marie-Tooth type 4C neuropathy (KIAA1985) (Senderek et al. Senderek et al., 2003Senderek J Bergmann C Stendel C Kirfel J Verpoorten N De Jonghe P Timmerman V et al.Mutations in a gene encoding a novel SH3/TPR domain protein cause autosomal recessive Charcot-Marie-Tooth type 4C neuropathy.Am J Hum Genet. 2003; 73: 1106-1119Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Since mutations in KIAA1279 are associated with HSCR and PMG, its protein product may play a pivotal role in both peripheral and central nervous system development. PMG is traditionally classified as a result of a disruption such as fetal hypoxic ischemic damage, infection, or exposure to toxic drugs in the second trimester (13–21 wk) (Barth and van der Harten Barth and van der Harten, 1985Barth PG van der Harten JJ Parabiotic twin syndrome with topical isocortical disruption and gastroschisis.Acta Neuropathol. 1985; 67: 345-349Crossref PubMed Scopus (53) Google Scholar; Barth Barth, 2003Barth PG Fetal disruption as a cause of neuronal migration defects.in: Barth PG Disorders of neuronal migration. MacKeith Press, London2003: 182-194Google Scholar). However, the identification of KIAA1279 as the GOSHS gene underlines the importance of genetic factors in the etiology of this neuronal migration disorder of the cortex, as was already proven (Mitchell et al. Mitchell et al., 2003Mitchell TN Free SL Williamson KA Stevens JM Churchill AJ Hanson IM Shorvon SD Moore AT van Heyningen V Sisodiya SM Mitchell TN Polymicrogyria and absence of pineal gland due to PAX6 mutation.Ann Neurol. 2003; 53: 658-663Crossref PubMed Scopus (133) Google Scholar; Piao et al. Piao et al., 2004Piao X Hill RS Bodell A Chang BS Basel-Vanagaite L Straussberg R Dobyns WB Qasrawi B Winter RM Innes AM Voit T Ross ME Michaud JL Descarie JC Barkovich AJ Walsh CA G protein-coupled receptor-dependent development of human frontal cortex.Science. 2004; 303: 2033-2036Crossref PubMed Scopus (416) Google Scholar) or suggested (Ciardo et al. Ciardo et al., 2001Ciardo F Zamponi N Specchio N Parmeggiani R Guerrini R Autosomal recessive polymicrogyria with infantile spasms and limb deformities.Neuropediatrics. 2001; 32: 325-329PubMed Google Scholar; Chang et al. Chang et al., 2004Chang BS Piao X Giannini C Cascino GD Scheffer I Woods CG Topcu M Tezcan K Bodell A Leventer RJ Barkovich AJ Grant PE Walsh CA Bilateral generalized polymicrogyria (BGP): a distinct syndrome of cortical malformation.Neurology. 2004; 62: 1722-1728Crossref PubMed Scopus (58) Google Scholar) for other forms of PMG. Under the assumption that the main function of KIAA1279 is to bind other proteins, these target peptides might be (known) HSCR- or PMG-associated susceptibility factors. More likely, however, these targets might play a more general role in neuronal development, since a malformation of cortical organization is a constant feature found in the Moroccan family, whereas HSCR is a variable feature. Elucidation of KIAA1279 function and, more important, studies of KIAA1279 expression and protein interactions may provide new insight into the molecular basis of PMG and HSCR. We thank the patients and their families, for their support and their willingness to donate samples; Jeannette Hoogeboom and Martijn Breuning, for patient care; Jim Barkovich, for help with classification of PMG; Martinus Niermeijer, for his guidance; Elisabeth Lodder and Peter van der Spek, for bioinformatics support; Herma van der Linde and Erik Simons, for technical assistance; and Tom de Vries Lentsch, for help with preparation of the figures. This work was funded, in part, by Nederlandse organisatie voor Wetenschappelijk Onderzoek grants 901-04-210 and 901-04-225 (to R.M.W.H.).

The KE family is a large three-generation pedigree in which half the members are affected with a severe speech and language disorder that is transmitted as an autosomal dominant monogenic trait. In previously published work, we localized the gene responsible (SPCH1) to a 5.6-cM region of 7q31 between D7S2459 and D7S643. In the present study, we have employed bioinformatic analyses to assemble a detailed BAC-/PAC-based sequence map of this interval, containing 152 sequence tagged sites (STSs), 20 known genes, and >7.75 Mb of completed genomic sequence. We screened the affected chromosome 7 from the KE family with 120 of these STSs (average spacing <100 kb), but we did not detect any evidence of a microdeletion. Novel polymorphic markers were generated from the sequence and were used to further localize critical recombination breakpoints in the KE family. This allowed refinement of the SPCH1 interval to a region between new markers 013A and 330B, containing approximately 6.1 Mb of completed sequence. In addition, we have studied two unrelated patients with a similar speech and language disorder, who have de novo translocations involving 7q31. Fluorescence in situ hybridization analyses with BACs/PACs from the sequence map localized the t(5;7)(q22;q31.2) breakpoint in the first patient (CS) to a single clone within the newly refined SPCH1 interval. This clone contains the CAGH44 gene, which encodes a brain-expressed protein containing a large polyglutamine stretch. However, we found that the t(2;7)(p23;q31.3) breakpoint in the second patient (BRD) resides within a BAC clone mapping >3.7 Mb distal to this, outside the current SPCH1 critical interval. Finally, we investigated the CAGH44 gene in affected individuals of the KE family, but we found no mutations in the currently known coding sequence. These studies represent further steps toward the isolation of the first gene to be implicated in the development of speech and language.

Elastin is the protein responsible for the characteristic elastic properties of many tissues including the skin, lungs and large blood vessels. Loss-of-function mutations in the elastin gene are known to cause the heart defect supravalvular aortic stenosis (SVAS), We and others have identified deletions, nonsense mutations and splice site mutations in SVAS patients that abolish the function of one elastin gene. We have now identified an elastin mutation in a patient with a completely different phenotype, the rare autosomal dominant condition cutis laxa. A frameshift mutation in exon 32 of the elastin gene is predicted to replace 37 amino acids at the C-terminus of elastin by a novel sequence of 62 amino acids. mRNA and immunoprecipitation studies show that the mutant allele is expressed. Electron microscopy of skin sections shows abnormal branching and fragmentation in the amorphous elastin component, and immunocytochemistry shows reduced elastin deposition in the elastic fibres and fewer microfibrils in the dermis. These findings suggest that the mutant tropoelastin protein is synthesized, secreted and incorporated into the elastic matrix, where it alters the architecture of elastic fibres, Interference with cross-linking would reduce elastic recoil in affected tissues and explain the cutis laxa phenotype.

We describe the complete exon-intron structure of the human elastin (ELN) gene located at chromosome 7q11.23. There are 34 exons occupying approximately 47 kb of genomic DNA. All exons are in-frame, allowing exon skipping without disrupting the reading frame. Microsatellites are located in introns 17 and 18. Deletions of all or large parts of the ELN gene have been previously reported in two patients with supravalvular aortic stenosis (SVAS), and SVAS is also a frequent feature of Williams syndrome, where patients are hemizygous for ELN. We list primer pairs for amplifying each exon, with flanking intron, from genomic DNA to allow detection of point mutations in the ELN gene. We show that some patients with isolated SVAS have point mutations that are predicted to lead to premature chain termination. Knowledge of the genomic structure will allow more extensive mutation screening in genomic DNA of patients with SVAS and other conditions.

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.

Many syndromes are named in an eponymous fashion after the original authors, or occasionally after the original patients, but the name of this syndrome is derived from the hospitals where the first two patients were described.In 1973 Pelletier and Feingold' described a boy seen at the Boston Floating Hospital with short stature, delayed speech development, and a striking facial appearance.A further report of a similar case was published in 1974 by Leisti et al.2 Their patient attended the Harbor General Hospital, Torrance, California.They suggested the term Floating-Harbor syndrome, which for its memorable quality is likely to become established.