Charles Shaw-Smith

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features

The underlying causes of learning disability and dysmorphic features in many patients remain unidentified despite extensive investigation. Routine karyotype analysis is not sensitive enough to detect subtle chromosome rearrangements (less than 5 Mb). The presence of subtle DNA copy number changes was investigated by array-CGH in 50 patients with learning disability and dysmorphism, employing a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Twelve copy number abnormalities were identified in 12 patients (24% of the total): seven deletions (six apparently de novo and one inherited from a phenotypically normal parent) and five duplications (one de novo and four inherited from phenotypically normal parents). Altered segments ranged in size from those involving a single clone to regions as large as 14 Mb. No recurrent deletion or duplication was identified within this cohort of patients. On the basis of these results, we anticipate that array-CGH will become a routine method of genome-wide screening for imbalanced rearrangements in children with learning disability.

Genomic and Genic Deletions of the FOX Gene Cluster on 16q24.1 and Inactivating Mutations of FOXF1 Cause Alveolar Capillary Dysplasia and Other Malformations

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.

Oesophageal atresia, tracheo-oesophageal fistula, and the VACTERL association: review of genetics and epidemiology

Oesophageal atresia and/or tracheo-oesophageal fistula are relatively common malformations occurring in approximately 1 in 3500 births. In around half of the cases (syndromic oesophageal atresia), there are associated anomalies, with cardiac malformations being the most common. In the remainder (non-syndromic cases), oesophageal atresia/tracheo-oesophageal fistula occur in isolation. Data from twin and family studies suggest that genetic factors do not play a major role, and yet there are well-defined instances of this malformation where genetic factors clearly are important. This is highlighted by the recent identification of no fewer than three separate genes with a role in the aetiology of oesophageal atresia: those for Feingold syndrome (N-MYC), anophthalmia-oesophageal-genital (AEG) syndrome (SOX2), and CHARGE syndrome (CHD7). Additional support for genetic factors in this malformation comes from chromosomal studies and mouse models. This paper reviews current knowledge of the genetics and epidemiology of the different oesophageal atresia/tracheo-oesophageal fistula syndromes and associations.

GATA6 haploinsufficiency causes pancreatic agenesis in humans

Understanding the regulation of pancreatic development is key for efforts to develop new regenerative therapeutic approaches for diabetes. Rare mutations in PDX1 and PTF1A can cause pancreatic agenesis, however, most instances of this disorder are of unknown origin. We report de novo heterozygous inactivating mutations in GATA6 in 15/27 (56%) individuals with pancreatic agenesis. These findings define the most common cause of human pancreatic agenesis and establish a key role for the transcription factor GATA6 in human pancreatic development.

Prenatal detection of unbalanced chromosomal rearrangements by array CGH

Background: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. Methods: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. Results: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. Conclusions: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.

Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ∼400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease.

DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

Genetic factors in esophageal atresia, tracheo-esophageal fistula and the VACTERL association: Roles for FOXF1 and the 16q24.1 FOX transcription factor gene cluster, and review of the literature

Esophageal atresia with/without tracheo-esophageal fistula is a relatively common malformation, occurring in around 1 in 3500 births. In around half of cases, additional malformations are present, forming either a syndrome of known genetic aetiology, or a recognised association, of which the VACTERL association (Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo-Esophageal fistula, Renal and Limb malformations) is the most recognised. Recently, microdeletions of the FOX gene cluster at 16q24.1, comprising four genes, FOXF1, MTHFSD, FOXC2 and FOXL1, were reported to cause a phenotype resembling VACTERL association, with vertebral anomalies, gastro-intestinal atresias (esophageal, duodenal and anal), congenital heart malformations, and urinary tract malformations, as well as a rare lethal developmental anomaly of the lung, alveolar capillary dysplasia. This article reviews these new data alongside other genetic causes of syndromic esophageal atresia, and also highlights information from relevant mouse models, particularly those for genes in the Sonic Hedgehog pathway.

High incidence of recurrent copy number variants in patients with isolated and syndromic Müllerian aplasia

Background Congenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer–Rokitansky–Kuster–Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially syndromic cases, are caused by variation in copy number at different genomic loci. Methods In order to obtain an overview of the contribution of copy number variation to both isolated and syndromic forms of Müllerian aplasia, copy number assays were performed in a series of 63 cases, of which 25 were syndromic and 38 isolated. Results A high incidence (9/63, 14%) of recurrent copy number variants in this cohort is reported here. These comprised four cases of microdeletion at 16p11.2, an autism susceptibility locus not previously associated with Müllerian aplasia, four cases of microdeletion at 17q12, and one case of a distal 22q11.2 microdeletion. Microdeletions at 16p11.2 and 17q12 were found in 4/38 (10.5%) cases with isolated Müllerian aplasia, and at 16p11.2, 17q12 and 22q11.2 (distal) in 5/25 cases (20%) with syndromic Müllerian aplasia. Conclusion The finding of microdeletion at 16p11.2 in 2/38 (5%) of isolated and 2/25 (8%) of syndromic cases suggests a significant contribution of this copy number variant alone to the pathogenesis of Müllerian aplasia. Overall, the high incidence of recurrent copy number variants in all forms of Müllerian aplasia has implications for the understanding of the aetiopathogenesis of the condition, and for genetic counselling in families affected by it.