Rodger Palmer

Over- and Underdosage of SOX3 Is Associated with Infundibular Hypoplasia and Hypopituitarism

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS

Congenital melanocytic nevi (CMN) can be associated with neurological abnormalities and an increased risk of melanoma. Mutations in NRAS, BRAF, and Tp53 have been described in individual CMN samples; however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesized that a single postzygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and nonmelanocytic central nervous system (CNS) lesions. From 15 patients, 55 samples with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12 out of 15 patients, but were absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In 10 patients, the mutation was consistently c.181C>A, p.Q61K, and in 2 patients c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from 5 patients were mutation positive, despite NRAS rarely being reported as mutated in CNS tumors. Loss of heterozygosity was associated with the onset of melanoma in two cases, implying a multistep progression to malignancy. These results suggest that single postzygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.

SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

CONTEXT Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathkes pouch, the developing anterior pituitary, and the eye. CONCLUSIONS Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

Improving diagnosis and broadening the phenotypes in early-onset seizure and severe developmental delay disorders through gene panel analysis

Background We sought to investigate the diagnostic yield and mutation spectrum in previously reported genes for early-onset epilepsy and disorders of severe developmental delay. Methods In 400 patients with these disorders with no known underlying aetiology and no major structural brain anomaly, we analysed 46 genes using a combination of targeted sequencing on an Illumina MiSeq platform and targeted, exon-level microarray copy number analysis. Results We identified causative mutations in 71/400 patients (18%). The diagnostic rate was highest among those with seizure onset within the first two months of life (39%), although overall it was similar in those with and without seizures. The most frequently mutated gene was SCN2A (11 patients, 3%). Other recurrently mutated genes included CDKL5, KCNQ2, SCN8A (six patients each), FOXG1, MECP2, SCN1A, STXBP1 (five patients each), KCNT1, PCDH19, TCF4 (three patients each) and ATP1A3, PRRT2 and SLC9A6 (two patients each). Mutations in EHMT1, GABRB3, LGI1, MBD5, PIGA, UBE3A and ZEB2 were each found in single patients. We found mutations in a number of genes in patients where either the electroclinical features or dysmorphic phenotypes were atypical for the identified gene. In only 11 cases (15%) had the clinician sufficient certainty to specify the mutated gene as the likely cause before testing. Conclusions Our data demonstrate the considerable utility of a gene panel approach in the diagnosis of patients with early-onset epilepsy and severe developmental delay disorders., They provide further insights into the phenotypic spectrum and genotype–phenotype correlations for a number of the causative genes and emphasise the value of exon-level copy number testing in their analysis.

Array comparative genomic hybridization: results from an adult population with drug-resistant epilepsy and co-morbidities.

Background The emergence of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated recognition of microdeletions and microduplications as risk factors for both generalised and focal epilepsies. Furthermore, there is evidence that some microdeletions/duplications, such as the 15q13.3 deletion predispose to a range of neuropsychiatric disorders, including intellectual disability (ID), autism, schizophrenia and epilepsy. We hypothesised that array CGH would reveal relevant findings in an adult patient group with epilepsy and complex phenotypes. Methods 82 patients (54 from the National Hospital for Neurology and Neurosurgery and 28 from King’s College Hospital) with drug-resistant epilepsy and co-morbidities had array CGH. Separate clinicians ordered array CGH and separate platforms were used at the two sites. Results In the two independent groups we identified copy number variants judged to be of pathogenic significance in 13.5% (7/52) and 20% (5/25) respectively, noting that slightly different selection criteria were used, giving an overall yield of 15.6%. Sixty-nine variants of unknown significance were also identified in the group from the National Hospital for Neurology and Neurosurgery and 5 from the King’s College Hospital patient group. Conclusion We conclude that array CGH be considered an important investigation in adults with complicated epilepsy and, at least at present for selected patients, should join the diagnostic repertoire of clinical history and examination, neuroimaging, electroencephalography and other indicated investigations in generating a more complete formulation of an individual’s epilepsy.

Sacral dysgenesis associated with terminal deletion of chromosome 7q: a report of two families.

Abstract Most cases of sacral dysgenesis are considered to be sporadic events. We present two families in whom the presence of associated clinical features prompted specific investigation of chromosome 7, leading to the identification of an underlying chromosome 7q deletion causing sacral dysgenesis. All affected individuals had microcephaly and developmental delay. Detailed cytogenetic studies confirmed that all three affected individuals had a deletion of chromosome 7q associated with their sacral dysgenesis, developmental delay and related problems. The three affected patients were studied clinically, radiologically and cytogenetically. Eleven unaffected individuals from the two families were also investigated by genetic studies, specifically evaluating chromosome 7. Conclusion It is important that detailed family history, evaluation of associated malformations and the overall clinical picture be considered in identifying the underlying diagnosis in cases of anal stenosis/sacral agenesis. The cases we present demonstrate the value of detailed chromosome studies in such situations.

Heterogeneity of mutational mechanisms and modes of inheritance in auriculocondylar syndrome

Background Auriculocondylar syndrome (ACS) is a rare craniofacial disorder consisting of micrognathia, mandibular condyle hypoplasia and a specific malformation of the ear at the junction between the lobe and helix. Missense heterozygous mutations in the phospholipase C, β 4 (PLCB4) and guanine nucleotide binding protein (G protein), α inhibiting activity polypeptide 3 (GNAI3) genes have recently been identified in ACS patients by exome sequencing. These genes are predicted to function within the G protein-coupled endothelin receptor pathway during craniofacial development. Results We report eight additional cases ascribed to PLCB4 or GNAI3 gene lesions, comprising six heterozygous PLCB4 missense mutations, one heterozygous GNAI3 missense mutation and one homozygous PLCB4 intragenic deletion. Certain residues represent mutational hotspots; of the total of 11 ACS PLCB4 missense mutations now described, five disrupt Arg621 and two disrupt Asp360. The narrow distribution of mutations within protein space suggests that the mutations may result in dominantly interfering proteins, rather than haploinsufficiency. The consanguineous parents of the patient with a homozygous PLCB4 deletion each harboured the heterozygous deletion, but did not present the ACS phenotype, further suggesting that ACS is not caused by PLCB4 haploinsufficiency. In addition to ACS, the patient harbouring a homozygous deletion presented with central apnoea, a phenotype that has not been previously reported in ACS patients. Conclusions These findings indicate that ACS is not only genetically heterogeneous but also an autosomal dominant or recessive condition according to the nature of the PLCB4 gene lesion.

A novel 5q11.2 deletion detected by microarray comparative genomic hybridisation in a child referred as a case of suspected 22q11 deletion syndrome

The 22q11 deletion syndrome (22q11DS) is a developmental syndrome comprising of heart, palate, thymus and parathyroid glands defects. Individuals with 22q11DS usually carry a 1.5- to 3-Mb heterozygous deletion on chromosome 22q11.2. However, there are many patients with features of 22q11DS without a known cause from conventional karyotype and FISH analysis. Six patients with features of 22q11DS, a normal chromosomal and FISH 22q11 analysis, were selected for investigation by microarray genomic comparative hybridisation (array CGH). Array-CGH is a powerful technology enabling detection of submicroscopic chromosome duplications and deletions by comparing a differentially labelled test sample to a control. The samples are co-hybridised to a microarray containing genomic clones and the resulting ratio of fluorescence intensities on each array element is proportional to the DNA copy number difference. No chromosomal changes were detected by hybridisation to a high resolution array representing chromosome 22q. However, one patient was found to have a 6-Mb deletion on 5q11.2 detected by a whole genome 1-Mb array. This deletion was confirmed with fluorescence in-situ hybridisation (FISH) and microsatellite marker analysis. It is the first deletion described in this region. The patient had tetralogy of Fallot, a bifid uvula and velopharyngeal insufficiency, short stature, learning and behavioural difficulties. This case shows the increased sensitivity of array CGH over detailed karyotype analysis for detection of chromosomal changes. It is anticipated that array CGH will improve the clinician’s capacity to diagnose congenital syndromes with an unknown aetiology.

Sotos syndrome, infantile hypercalcemia, and nephrocalcinosis: a contiguous gene syndrome

Sotos syndrome is characterized by overgrowth, a typical facial appearance, and learning difficulties. It is caused by heterozygous mutations, including deletions, of NSD1 located at chromosome 5q35. Here we report two unrelated cases of Sotos syndrome associated with nephrocalcinosis. One patient also had idiopathic infantile hypercalcemia. Genetic investigations revealed heterozygous deletions at 5q35 in both patients, encompassing NSD1 and SLC34A1 (NaPi2a). Mutations in SLC34A1 have previously been associated with hypercalciuria/nephrolithiasis. Our cases suggest a contiguous gene deletion syndrome including NSD1 and SLC34A1 and provide a potential genetic basis for idiopathic infantile hypercalcemia.

Young-onset parkinsonism due to homozygous duplication of α-synuclein in a consanguineous family†‡§¶

With a growing list of monogenetic causes of Parkinsons disease (PD),1–3 prioritization of genetic testing is made on the presumed mode of inheritance. In patients with early-onset PD from consanguineous families, the most common mode of inheritance is autosomal recessive. Rarely, an individual may be homozygous for a mutation inherited in an autosomal dominant manner, complicating the diagnostic process. The proband is a 36-year-old female from a consanguineous Pakistani family (Fig. 1A). She presented with a 5-year history of left-sided stiffness, micrographia, festinant gait, tremor, and falls. Past medical history included postpartum psychosis and severe depression. There were no symptoms of autonomic dysfunction. Neurological examination at the age of 35 years revealed severe bilateral bradykinesia, rigidity, worse on the left, and mild bilateral upper limb rest tremor. Gait was shuffling, with freezing and complete loss of postural reflexes. Bilateral ankle clonus was noted. Initially, there was a poor response to dopamine agonists. She responded well to 100/25 mg co-careldopa 3 times per day, but developed wearing offs and peak-dose dyskinesias after 4 months of treatment. There was no family history of parkinsonism. The probands mothers neurological examination at the age of 72 years was normal. The probands father, who died at age 64 years from a stroke, had no symptoms or signs of parkinsonism as described by his family. Her siblings and children were asymptomatic, but were unavailable for examination. Figure 1 A: Pedigree and clinical information on the family. A half filled symbol represents the proband (arrow), who is the only individual affected by parkinsonism. Clinically unaffected but confirmed mutation carrier (the probands mother) is represented by ... Investigations in the proband included routine blood work, copper studies, and white cell enzymes, and were all normal. Dopamine transporter scan (DAT-SCAN) showed bilateral reduction in tracer uptake in the striatum, more significant on the right. Formal neuropsychological examination revealed a mild degree of intellectual under-functioning from premorbid estimates, with anterior/subcortical dysfunction. Mutations in PARKIN, PINK1, and common LRRK2 mutations were excluded. Multiplex ligation-dependent probe amplification (MLPA) showed 4 copies of SNCA (Fig. 1B). Comparative genomic hybridization (Nimblegen 135 K array Roche-Niblegen) showed copy number gain of 0.928 Mb, containing 5 genes including SNCA (Fig. 1C). Single-nucleotide polymorphism (SNP) array showed this to be homozygous. Fluorescent in situ-hybridization demonstrated duplication of SNCA on both alleles (Supplemental Figure 1). The probands mothers MLPA showed SNCA duplication. Considering the probands homozygosity for SNCA duplication, her father is likely to have had an SNCA duplication. Less likely, de novo duplication (in addition to duplication inherited from mother) occurred in the proband. SNCA multiplication is a rare cause of autosomal dominant PD.4,5 SNCA triplications (4 copies of SNCA) are fully penetrant; with early-onset and rapidly progressive parkinsonism associated with dementia, autonomic dysfunction, and psychiatric features.4 With SNCA duplications (3 SNCA copies), the disease clinically resembles idiopathic PD5 and reduced penetrance is recognized.6 Our patient has 4 SNCA copies and her clinical phenotype closely matches that of patients with SNCA triplications4 and is in keeping with the single previously described case of SNCA double duplication.7 Homozygous SNCA duplications are a rare cause of young-onset parkinsonism and should be considered in the patients with a family history compatible with recessive inheritance. Early cognitive dysfunction and depression are consistently reported in patients with 4 SNCA copies and may be regarded as “red flags” for SNCA multiplications.