Emma McCann

Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis

Background: Schwannomatosis is a rare condition characterised by multiple schwannomas and lack of involvement of the vestibular nerve. A recent report identified bi-allelic mutations in the SMARCB1/INI1 gene in a single family with schwannomatosis. We aimed to establish the contribution of the SMARCB1 and the NF2 genes to sporadic and familial schwannomatosis in our cohort. Methods: We performed DNA sequence and dosage analysis of SMARCB1 and NF2 in 28 sporadic cases and 15 families with schwannomatosis. Results: We identified germline mutations in SMARCB1 in 5 of 15 (33.3%) families with schwannomatosis and 2 of 28 (7.1%) individuals with sporadic schwannomatosis. In all individuals with a germline mutation in SMARCB1 in whom tumour tissue was available, we detected a second hit with loss of SMARCB1. In addition, in all affected individuals with SMARCB1 mutations and available tumour tissue, we detected bi-allelic somatic inactivation of the NF2 gene. SMARCB1 mutations were associated with a higher number of spinal tumours in patients with a positive family history (p = 0.004). Conclusion: In contrast to the recent report where no NF2 mutations were identified in a schwannomatosis family with SMARCB1 mutations, in our cohort, a four hit model with mutations in both SMARCB1 and NF2 define a subset of patients with schwannomatosis.

Clinical and cytogenetic characterization of 13 Dutch patients with deletion 9p syndrome: Delineation of the critical region for a consensus phenotype†

The deletion 9p syndrome is caused by a constitutional monosomy of part of the short arm of chromosome 9. It is clinically characterized by dysmorphic facial features (trigonocephaly, midface hypoplasia, and long philtrum), hypotonia and mental retardation. Deletion 9p is known to be heterogeneous and exhibits variable deletion sizes. The critical region for a consensus phenotype has been reported to be located within a ∼4–6 Mb interval on 9p22. In the present study, deletion breakpoints were determined in 13 Dutch patients by applying fluorescence in situ hybridization (FISH) and in some specific cases by array‐based comparative genomic hybridization (array CGH). No clear genotype–phenotype correlation could be established for various developmental features. However, we were able to narrow down the critical region for deletion 9p syndrome to ∼300 kb. A functional candidate gene for trigonocephaly, the CER1 gene, appeared to be located just outside this region. Sequence analysis of this gene in nine additional patients with isolated trigonocephaly did not reveal any pathogenic mutations.

WT1 mutations in Meacham syndrome suggest a coelomic mesothelial origin of the cardiac and diaphragmatic malformations

Meacham syndrome is a rare sporadically occurring multiple malformation syndrome characterized by male pseudohermaphroditism with abnormal internal female genitalia comprising a uterus and double or septate vagina, complex congenital heart defect and diaphragmatic abnormalities. We report on eight new cases of this condition, two of whom were shown to have heterozygous missense mutations in the C‐terminal zinc finger domains of WT1: Arg366Cys and Arg394Trp. These data represent clinical and molecular evidence that the WT1 gene plays a central role in normal development of the diaphragm and the proepicardially derived tissues. Identification of WT1 expression in the region of coelomic mesothelium which will form the proepicardium and diaphragm provides a plausible unifying patterning defect in these cases. Interestingly, the Arg366Cys mutation has been previously reported in Denys–Drash syndrome and Arg394Trp mutation has been previously reported in both isolated Wilms tumor and Denys–Drash syndrome. This phenotypic diversity with a single mutation suggests there are other factors modulating all aspects of WT1 function during human development. If genetic modifiers of WT1 can be identified in animal models these become good candidate genes for the cases with Meacham syndrome we report on here where WT1 mutations cannot be identified.

VSX2 in microphthalmia: a novel splice site mutation producing a severe microphthalmia phenotype

Microphthalmia shows great genetic and clinical heterogeneity, whether as part of a syndrome or an isolated ocular phenotype. Chromosomal or single-gene disorders and teratogens may all cause microphthalmia. Associated syndromic features include cardiac problems, clefting, microcephaly and learning disabilities.1 Microphthalmia is frequently bilateral, but commonly asymmetrical in severity. Homozygous mutations in VSX2/CHX10 have been demonstrated in human and murine microphthalmia.2 3 VSX2 is thought to act principally as a repressor of transcription, particularly of the genes encoding cyclin-dependent kinase inhibitor (p27kip1) and microphthalmia transcription factor (MITF).4 These repressive roles enable cell proliferation by preventing retinal progenitor cells from exiting the cell cycle, and by maintaining neuroretinal cell identity. Loss of these functions therefore causes failures in eye development. Other genes implicated in microphthalmia include SOX2 , PAX6 , sonic hedgehog ( SHH ), RAX , OTX2 , CRYBA …

Novel phenotype of craniosynostosis and ocular anterior chamber dysgenesis with a fibroblast growth factor receptor 2 mutation

Fibroblast growth factor receptor 2 (FGFR2) mutations are associated with syndromic and non‐syndromic craniosynostoses. More recently it has been recognized that FGFR2 may have a role in the development of the anterior chamber of the eye following the finding of a specific FGFR2 mutation (p.Ser351Cys, c.1231 C → G) with anterior chamber dysgenesis. Affected patients had a severe craniofacial phenotype and clinical course. A child with a different FGFR2 mutation (p.Ala344Ala, c1032 G → A heterozygote), premature fusion of the sagittal suture, and an Axenfeld–Rieger anomaly but otherwise normal clinical course is reported. The case provides further evidence that FGFR2 has a role in anterior chamber embryogenesis.

A review of prenatally detected femoral abnormalities.

Antenatally detected femoral abnormalities are a rare finding, and form a heterogeneous group in terms of diagnosis and prognosis. The objective of this study is to evaluate the management and outcome of fetuses after prenatal detection of femoral abnormalities in a tertiary care Fetal Medicine Unit over a 5-year period, thus facilitating accurate diagnosis and appropriate counseling. Patients were identified through a Regional Fetal Anomaly Database or clinician recall. Information was collected on whether the abnormality was an isolated or generalized skeletal abnormality, whether nonskeletal abnormalities were detected, what prognosis was counselled, whether referral to clinical genetics occurred, what investigations were performed, what was the eventual outcome and whether a final diagnosis was reached. A definitive diagnosis was made in 68% (28 of 41) of cases. However, a correct diagnosis was only achieved in 19% (eight of 41) of cases before delivery. Prognosis was correctly predicted in 83% (34 of 41) of cases. Families found the degree of uncertainty difficult, with 67% (six of nine) of patients where the prognosis was ‘uncertain’ electing to terminate the pregnancy. Eighty percent (eight of 10) of those where the condition was deemed ‘lethal’ also elected to end the pregnancy. In conclusion, it is difficult to achieve a correct diagnosis before delivery when a femoral abnormality is identified; however, detailed antenatal assessment may aid the differential diagnosis and help to determine prognosis. This was more likely to be achieved if the patient was seen in association with a clinical geneticist. A multidisciplinary team approach involving fetal medicine, genetics, paediatric radiology, and pathology is advocated. Fetal examination, medical photography, chromosomes and DNA storage, postnatal radiography and postmortem facilitate postnatal diagnosis and counseling.

Congenital melanocytic naevus with associated neurofibroma and schwannoma-like change.

Congenital melanocytic naevus and neurofibromatosis type 1 are distinct clinical entities. A diagnosis of neurofibromatosis is difficult to make in the presence of a congenital melanocytic naevus because nodules may arise in the naevus that have similar histopathological appearances to neurofibromata. A case is reported where nodules arising from a naevus were examined histologically and were found to have neurofibroma and schwannoma like elements but strong positivity for S100 protein in keeping with dermal melanocytes. Lisch nodules were also said to be found in the patient but may represent nodular naevi of the irides. It is important that histopathological findings are interpreted within a clinical context and S100 protein immunohistochemical stain is valuable in helping to differentiate these two conditions.

A family with Duane anomaly and distal limb abnormalities: a further family with the arthrogryposis-ophthalmoplegia syndrome.

A two‐generation family is reported in which three members have Duane anomaly and distal limb abnormalities. All three affected have photopic electroretinogram responses that are abnormal or at the lower limit of the normal range with normal scotopic responses. Two affected family members also have hearing loss. The likeliest diagnosis is the syndrome listed as “arthrogryposis‐ophthalmoplegia syndrome” on the London Dysmorphology Database or as “arthrogryposis with oculomotor limitation and electroretinal abnormalities” or “oculomelic aplasia” in OMIM [MIM 108145]. In view of the similarities with Okihiro syndrome, a search for mutations within the SALL4 gene was undertaken, but none were identified.

The contribution of non-canonical splicing mutations to severe dominant developmental disorders

Mutations which perturb normal pre-mRNA splicing are significant contributors to human disease. We used exome sequencing data from 7,833 probands with developmental disorders (DD) and their unaffected parents, as well as >60,000 aggregated exomes from the Exome Aggregation Consortium, to investigate selection around the splice site, and quantify the contribution of splicing mutations to DDs. Patterns of purifying selection, a deficit of variants in highly constrained genes in healthy subjects and excess de novo mutations in patients highlighted particular positions within and around the consensus splice site of greater functional relevance. Using mutational burden analyses in this large cohort of proband-parent trios, we could estimate in an unbiased manner the relative contributions of mutations at canonical dinucleotides (73%) and flanking non-canonical positions (27%), and calculated the positive predictive value of pathogenicity for different classes of mutations. We identified 18 patients with likely diagnostic de novo mutations in dominant DD-associated genes at non-canonical positions in splice sites. We estimate 35-40% of pathogenic variants in non-canonical splice site positions are missing from public databases.Accurate and efficient pre-mRNA splicing is crucial for normal development and function, and mutations which perturb normal splicing patterns are significant contributors to human disease. We used exome sequencing data from 7,833 probands with developmental disorders (DD) and their unaffected parents to quantify the contribution of splicing mutations to DDs. Patterns of purifying selection, a deficit of variants in highly constrained genes in healthy subjects and excess de novo mutations in patients highlighted particular positions within and around the consensus splice site of greater disease relevance. Using mutational burden analyses in this large cohort of proband-parent trios, we could estimate in an unbiased manner the relative contributions of mutations at canonical dinucleotides (73%) and flanking non-canonical positions (27%), and calculated the positive predictive value of pathogenicity for different classes of mutations. We identified 18 likely diagnostic de novo mutations in dominant DD-associated genes at non-canonical positions in splice sites. We estimate 35-40% of pathogenic variants in non-canonical splice site positions are missing from public databases.