Kathleen A. Williamson

Heterozygous mutations of OTX2 cause severe ocular malformations

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.

PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans.

PAX6 is widely expressed in the central nervous system. Heterozygous PAX6 mutations in human aniridia cause defects that would seem to be confined to the eye. Magnetic resonance imaging (MRI) and smell testing reveal the absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of aniridia cases, which shows that PAX6 haploinsuffiency causes more widespread human neuro developmental anomalies.

SOX2 anophthalmia syndrome

Heterozygous, de novo, loss‐of‐function mutations in SOX2 have been shown to cause bilateral anophthalmia. Here we provide a detailed description of the clinical features associated with SOX2 mutations in the five individuals with reported mutations and four newly identified cases (including the first reported SOX2 missense mutation). The SOX2‐associated ocular malformations are variable in type, but most often bilateral and severe. Of the nine patients, six had bilateral anophthalmia and two had anophthalmia with contralateral microphthalmia with sclerocornea. The remaining case had anophthalmia with contralateral microphthalmia, posterior cortical cataract and a dysplastic optic disc, and was the only patient to have measurable visual acuity. The relatively consistent extraocular phenotype observed includes: learning disability, seizures, brain malformation, specific motor abnormalities, male genital tract malformations, mild facial dysmorphism, and postnatal growth failure. Identifying SOX2 mutations from large cohorts of patients with structural eye defects has delineated a new, clinically‐recognizable, multisystem disorder and has provided important insight into the developmental pathways critical for morphogenesis of the eye, brain, and male genital tract.

Polymicrogyria and absence of pineal gland due to PAX6 mutation

Identification of genes involved in human cerebral development is important for our understanding of disorders with potential neurodevelopmental causes such as epilepsy and learning disability. Murine models suggest that PAX6 plays a key role in human brain development. With magnetic resonance imaging in 24 humans heterozygous for defined PAX6 mutations, we demonstrated widespread structural abnormalities including absence of the pineal gland and unilateral polymicrogyria. Ann Neurol 2003;53:658–663

Genetic analysis of chromosome 11p13 and the PAX6 gene in a series of 125 cases referred with aniridia.

A series of 125 patients referred primarily with aniridia classified as either sporadic (74), familial (24), or in association with WAGR syndrome (14) or other malformations (13) was analysed for mutations, initially by karyotyping and targeted FISH analysis of chromosome 11p13. These methods identified mutations in a significant proportion of patients, 34/125 (27%). Two cases had chromosome rearrangements involving 11p13, 16 cases had visible deletions, and 16 cases had cryptic deletions identified by FISH. The frequency of cryptic deletions in familial aniridia was 27% and in sporadic isolated aniridia was 22%. Of the 14 cases referred with WAGR syndrome, 10 (71%) had chromosomal deletions, 2 cryptic and 8 visible. Of the 13 cases with aniridia and other malformations, 5 (38%) had a chromosomal rearrangement or deletion. In 37 cases with no karyotypic or cryptic chromosome abnormality, sequence analysis of the PAX6 gene was performed. Mutations were identified in 33 cases; 22 with sporadic aniridia, 10 with familial aniridia and 1 with aniridia and other non‐WAGR syndrome associated anomalies. Overall, 67 of 71 cases (94%) undergoing full mutation analysis had a mutation in the PAX6 genomic region.

The genetic architecture of microphthalmia, anophthalmia and coloboma

Microphthalmia, anophthalmia and coloboma (MAC) are distinct phenotypes that represent a continuum of structural developmental eye defects. In severe bilateral cases (anophthalmia or severe microphthalmia) the genetic cause is now identifiable in approximately 80 percent of cases, with de novo heterozygous loss-of-function mutations in SOX2 or OTX2 being the most common. The genetic cause of other forms of MAC, in particular isolated coloboma, remains unknown in the majority of cases. This review will focus on MAC phenotypes that are associated with mutation of the genes SOX2, OTX2, PAX6, STRA6, ALDH1A3, RARB, VSX2, RAX, FOXE3, BMP4, BMP7, GDF3, GDF6, ABCB6, ATOH7, C12orf57, TENM3 (ODZ3), and VAX1. Recently reported mutation of the SALL2 and YAP1 genes are discussed in brief. Clinical and genetic features were reviewed in a total of 283 unrelated MAC cases or families that were mutation-positive from these 20 genes. Both the relative frequency of mutations in MAC cohort screens and the level of confidence in the assignment of disease-causing status were evaluated for each gene.

Genetic heterogeneity in Cornelia de Lange syndrome (CdLS) and CdLS-like phenotypes with observed and predicted levels of mosaicism

Background Cornelia de Lange syndrome (CdLS) is a multisystem disorder with distinctive facial appearance, intellectual disability and growth failure as prominent features. Most individuals with typical CdLS have de novo heterozygous loss-of-function mutations in NIPBL with mosaic individuals representing a significant proportion. Mutations in other cohesin components, SMC1A, SMC3, HDAC8 and RAD21 cause less typical CdLS. Methods We screened 163 affected individuals for coding region mutations in the known genes, 90 for genomic rearrangements, 19 for deep intronic variants in NIPBL and 5 had whole-exome sequencing. Results Pathogenic mutations [including mosaic changes] were identified in: NIPBL 46 [3] (28.2%); SMC1A 5 [1] (3.1%); SMC3 5 [1] (3.1%); HDAC8 6 [0] (3.6%) and RAD21 1 [0] (0.6%). One individual had a de novo 1.3 Mb deletion of 1p36.3. Another had a 520 kb duplication of 12q13.13 encompassing ESPL1, encoding separase, an enzyme that cleaves the cohesin ring. Three de novo mutations were identified in ANKRD11 demonstrating a phenotypic overlap with KBG syndrome. To estimate the number of undetected mosaic cases we used recursive partitioning to identify discriminating features in the NIPBL-positive subgroup. Filtering of the mutation-negative group on these features classified at least 18% as ‘NIPBL-like’. A computer composition of the average face of this NIPBL-like subgroup was also more typical in appearance than that of all others in the mutation-negative group supporting the existence of undetected mosaic cases. Conclusions Future diagnostic testing in ‘mutation-negative’ CdLS thus merits deeper sequencing of multiple DNA samples derived from different tissues.

Role of SOX2 mutations in human hippocampal malformations and epilepsy

Summary:  Purpose: Seizures are noted in a significant proportion of cases of de novo, heterozygous, loss‐of‐function mutations in SOX2, ascertained because of severe bilateral eye malformations. We wished to determine the underlying cerebral phenotype in SOX2 mutation and to test the candidacy of SOX2 as a gene contributing to human epilepsies.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Clinical and mutation analysis of 51 probands with anophthalmia and/or severe microphthalmia from a single center.

Clinical evaluation and mutation analysis was performed in 51 consecutive probands with severe eye malformations – anophthalmia and/or severe microphthalmia – seen in a single specialist ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions of SOX2, OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and genome‐wide array‐based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder. Two families with SOX2 mutations demonstrated a parent and child both with significant but highly variable eye malformations. Heterozygous loss‐of‐function mutations in SOX2 and OTX2 are the most common genetic pathology associated with severe eye malformations and bi‐allelic loss‐of‐function in STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations.