David J. Bunyan

Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification.

Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes.

Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome represent one condition with variable expression and age-related penetrance: results of a clinical study of PTEN mutation carriers

Background: The most commonly reported phenotypes described in patients with PTEN mutations are Bannayan–Riley–Ruvalcaba syndrome (BRRS), with childhood onset, macrocephaly, lipomas and developmental delay, and Cowden Syndrome (CS), an adult-onset condition recognised by mucocutaneous signs, with a risk of cancers, in particular those of the thyroid and breast. It has been suggested that BRRS and CS are the same condition, but the literature continues to separate them and seek a genotype–phenotype correlation. Objective: To study the clinical features of patients with known PTEN mutations and observe any genotype–phenotype correlation. Methods: In total, 42 people (25 probands and 17 non-probands) from 26 families of all ages with PTEN mutations were recruited through the UK clinical genetics services. A full clinical history and examination were undertaken. Results: We were unable to demonstrate a genotype–phenotype correlation. Furthermore, our findings in a 31-year-old woman with CS and an exon 1 deletion refutes previous reports that whole exon deletions are only found in patients with a BRRS phenotype. Conclusion: Careful phenotyping gives further support for the suggestion that BRRS and CS are actually one condition, presenting variably at different ages, as in other tumour-suppressor disorders such as neurofibromatosis type 1. This has important counselling implications, such as advice about cancer surveillance, for children diagnosed with BRRS.

Genotype-phenotype correlation in Smith-Magenis syndrome: evidence that multiple genes in 17p11.2 contribute to the clinical spectrum.

Purpose: Smith-Magenis syndrome (SMS) is a complex disorder that includes mental retardation, craniofacial and skeletal anomalies, and behavioral abnormalities. We report the molecular and genotype–phenotype analyses of 31 patients with SMS who carry 17p11.2 deletions or mutations in the RAI1 gene.Methods: Patients with SMS were evaluated by fluorescence in situ hybridization and/or sequencing of RAI1 to identify 17p11.2 deletions or intragenic mutations, respectively, and were compared for 30 characteristic features of this disorder by the Fisher exact test.Results: In our cohort, 8 of 31 individuals carried a common 3.5 Mb deletion, whereas 10 of 31 individuals carried smaller deletions, two individuals carried larger deletions, and one individual carried an atypical 17p11.2 deletion. Ten patients with nondeletion harbored a heterozygous mutation in RAI1. Phenotypic comparison between patients with deletions and patients with RAI1 mutations show that 21 of 30 SMS features are the result of haploinsufficiency of RAI1, whereas cardiac anomalies, speech and motor delay, hypotonia, short stature, and hearing loss are associated with 17p11.2 deletions rather than RAI1 mutations (P<.05). Further, patients with smaller deletions show features similar to those with RAI1 mutations.Conclusion: Although RAI1 is the primary gene responsible for most features of SMS, other genes within 17p11.2 contribute to the variable features and overall severity of the syndrome.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome

Recently, we identified 3′ end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele‐specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch‐like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2‐deficient families from multiple geographical origins with varying deletions all encompassing the 3′ end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu‐repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3′ end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics. Hum Mutat 32:1–8, 2011.

Novel heterozygous OTX2 mutations and whole gene deletions in anophthalmia, microphthalmia and coloboma†

Severe ocular malformations, including anophthalmia‐microphthalmia (AM), are responsible for around 25% of severe visual impairment in childhood. Recurrent interstitial deletions of 14q22–23 are associated with AM and a wide range of extra‐ocular phenotypes including brain anomalies. The homeobox gene OTX2 is located at 14q22.3 and has recently been identified as mutated in AM patients. Eight human OTX2 mutations have been reported in subjects with severe eye malformations, including AM, and variable developmental delay. We screened a novel AM cohort for mutations and deletions in OTX2, and identified four new mutations in six individuals and two cases of whole gene deletions. Our data suggest that OTX2 mutations and deletions account for 2–3% of AM cases.

8p23.1 duplication syndrome; a novel genomic condition with unexpected complexity revealed by array CGH

The 8p23.1 deletion syndrome is established but not an equivalent duplication syndrome. Here, we report five patients; a de novo prenatal case and two families in which 8p23.1 duplications have been directly transmitted from mothers to children. Dual-colour fluorescent in situ hybridisation, multiplex ligation-dependent probe amplification analysis and customised oligonucleotide array comparative genomic hybridisation (oaCGH) indicated an ∼3.75 Mb duplication of most of band 8p23.1 between the olfactory receptor/defensin repeats (ORDRs) in all cases. However, oaCGH revealed an additional duplication of 500 kb adjacent to the proximal ORDR in Family 1 and an additional deletion of 3.14 Mb within the Nablus Mask-Like Facial Syndrome region of 8q22.1 in Family 2. Copy number variation at introns 4–5 of the GATA4 gene was also identified. This 8p23.1 duplication syndrome is associated with a characteristic facial phenotype including a prominent forehead and arched eyebrows. Adrenal insufficiency, Tetralogy of Fallot, partial 2/3 syndactyly of the toes and cleft palate in some individuals may be explained by ascertainment bias, incomplete penetrance and/or the presence of the microdeletion in Family 2. The duplication is compatible with normal early childhood development but, although our adult cases live independent lives with varying degrees of support, learning difficulties have been experienced by some family members. We conclude that the 8p23.1 duplication syndrome is a genomic condition with an emerging but variable phenotype that may be under-diagnosed. Our results demonstrate that direct transmission does not distinguish genuine duplications from euchromatic variants and illustrate the power of array CGH to reveal unexpected additional imbalances in affected patients.

Gross rearrangements of the MECP2 gene are found in both classical and atypical Rett syndrome patients

MECP2 mutations are identifiable in ∼80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.

Epimutation of the TNDM locus and the Beckwith-Wiedemann syndrome centromeric locus in individuals with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is characterised by intra-uterine growth retardation, while Beckwith–Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome. Both TNDM and BWS may be caused by aberrant loss of methylation (LOM) at imprinted loci on chromosomes 6q24 and 11p15.5 respectively. Here we describe two patients with a clinical diagnosis of TNDM caused by LOM at the maternally methylated imprinted domain on 6q24; in addition, these patients had LOM at the centromeric differentially methylated region of 11p15.5. This shows that imprinting anomalies can affect more than one imprinted locus and may alter the clinical presentation of imprinted disease.

Transmitted duplication of 8p23.1–8p23.2 associated with speech delay, autism and learning difficulties

Duplications of distal 8p with and without significant clinical phenotypes have been reported and are often associated with an unusual degree of structural complexity. Here, we present a duplication of 8p23.1–8p23.2 ascertained in a child with speech delay and a diagnosis of ICD-10 autism. The same duplication was found in his mother who had epilepsy and learning problems. A combination of cytogenetic, FISH, microsatellite, MLPA and oaCGH analysis was used to show that the duplication extended over a minimum of 6.8 Mb between 3 539 893 and 10 323 426 bp. This interval contains 32 novel and 41 known genes, of which only microcephalin (MCPH1) is a plausible candidate gene for autism at present. The distal breakpoint of the duplicated region interrupts the CSMD1 gene in 8p23.2 and the medial breakpoint lies between the MSRA and RP1L1 genes in 8p23.1.An interchromosomal insertion between a normal and polymorphically inverted chromosome 8 is proposed to explain the origin of this duplication. Further mapped imbalances of distal 8p are needed to determine whether the autistic component of the phenotype in this family results from the cumulative imbalance of many genes or dosage imbalance of an individual susceptibility gene.

Further case of Rubinstein–Taybi syndrome due to a deletion in EP300

Rubinstein–Taybi syndrome (RSTS) is a heterogeneous disorder with approximately 45–55% of patients showing mutations in the CREB binding protein and a further 3% of patients having mutations in EP300. We report a male child with a deletion of exons 3–8 of the EP300 gene who has RSTS. He has a milder skeletal phenotype, a finding that has been described in other cases with EP300 mutations. The mother suffered from pre‐eclampsia and HELLP syndrome in the pregnancy. She subsequently developed a mullerian tumor of her cervix 6 years after the birth of her son.