Stefan J. White

Genetic heterogeneity in Rubinstein-Taybi syndrome: mutations in both the CBP and EP300 genes cause disease

CREB-binding protein and p300 function as transcriptional coactivators in the regulation of gene expression through various signal-transduction pathways. Both are potent histone acetyl transferases. A certain level of CREB-binding protein is essential for normal development, since inactivation of one allele causes Rubinstein-Taybi syndrome (RSTS). There is a direct link between loss of acetyl transferase activity and RSTS, which indicates that the disorder is caused by aberrant chromatin regulation. We screened the entire CREB-binding protein gene (CBP) for mutations in patients with RSTS by using methods that find point mutations and larger rearrangements. In 92 patients, we were able to identify a total of 36 mutations in CBP. By using multiple ligation-dependent probe amplification, we found not only several deletions but also the first reported intragenic duplication in a patient with RSTS. We extended the search for mutations to the EP300 gene and showed that mutations in EP300 also cause this disorder. These are the first mutations identified in EP300 for a congenital disorder.

Complex SNP-related sequence variation in segmental genome duplications.

There is uncertainty about the true nature of predicted single-nucleotide polymorphisms (SNPs) in segmental duplications (duplicons) and whether these markers genuinely exist at increased density as indicated in public databases. We explored these issues by genotyping 157 predicted SNPs in duplicons and control regions in normal diploid genomes and fully homozygous complete hydatidiform moles. Our data identified many true SNPs in duplicon regions and few paralogous sequence variants. Twenty-eight percent of the polymorphic duplicon sequences we tested involved multisite variation, a new type of polymorphism representing the sum of the signals from many individual duplicon copies that vary in sequence content due to duplication, deletion or gene conversion. Multisite variations can masquerade as normal SNPs when genotyped. Given that duplicons comprise at least 5% of the genome and many are yet to be annotated in the genome draft, effective strategies to identify multisite variation must be established and deployed.

Nine unknown rearrangements in 16p13.3 and 11p15.4 causing α- and β-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification

Background: Approximately 80% of the α- and 10% of the β-thalassaemias are caused by genomic deletions involving the α- and β-globin gene clusters on chromosomes 16p13.3 and 11p15.5, respectively. Gap-PCR, Southern blot analysis, and fluorescent in situ hybridisation are commonly used to identify these deletions; however, many deletions go undetected using conventional techniques. Methods: Patient samples for which no abnormalities had been found using conventional DNA techniques were analysed by a three colour multiplex ligation-dependent probe amplification assay. Two sets of 35 and 50 probes, covering a region of 700 kb of the α- and 500 kb of the β-globin gene cluster, respectively, were designed to detect rearrangements in the α- and β-globin gene clusters. Results: In 19 out of 38 patient samples, we found 11 different α-thalassaemia deletions, six of which were not previously described. Two novel deletions leaving the α-globin gene cluster intact were found to cause a complete downregulation of the downstream α-genes. Similarly, 31 out of 51 patient samples were found to carry 10 different deletions involving the β-globin gene cluster, three of which were not previously described. One involves the deletion of the locus control region leaving the β-globin gene cluster intact. Conclusions: These deletions, which are not easily detected by conventional techniques, may have clinical implications during pregnancy ranging from mild to life threatening microcytic haemolytic anaemia in neonates. The approach as described here is a rapid and sensitive method for high resolution analysis of the globin gene clusters and for any region of the genome.

Comprehensive Detection of Genomic Duplications and Deletions in the DMD Gene, by Use of Multiplex Amplifiable Probe Hybridization

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.

Identification of SOX3 as an XX male sex reversal gene in mice and humans

Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.

Peters Plus Syndrome Is Caused by Mutations in B3GALTL, a Putative Glycosyltransferase

Peters Plus syndrome is an autosomal recessive disorder characterized by anterior eye-chamber abnormalities, disproportionate short stature, and developmental delay. After detection of a microdeletion by array-based comparative genomic hybridization, we identified biallelic truncating mutations in the beta 1,3-galactosyltransferase-like gene (B3GALTL) in all 20 tested patients, showing that Peters Plus is a monogenic, primarily single-mutation syndrome. This finding is expected to put Peters Plus syndrome on the growing list of congenital malformation syndromes caused by glycosylation defects.

Deletion and duplication screening in the DMD gene using MLPA.

We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.

Mutations in MAP3K1 Cause 46,XY Disorders of Sex Development and Implicate a Common Signal Transduction Pathway in Human Testis Determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.

Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.

Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single‐strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non‐coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.

Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.