Genetic counselling and ethical issues with chromosome microarray analysis in prenatal testing.

Molecular karyotyping using chromosome microarray analysis (CMA) detects more pathogenic chromosomal anomalies than classical karyotyping, making CMA likely to become a first tier test for prenatal diagnosis. Detecting copy number variants of uncertain clinical significance raises ethical considerations. We consider the risk of harm to a woman or her fetus following the detection of a copy number variant of uncertain significance, whether it is ethically justifiable to withhold any test result information from a woman, what constitutes an 'informed choice' when women are offered CMA in pregnancy and whether clinicians are morally responsible for 'unnecessary' termination of pregnancy. Although we are cognisant of the distress associated with uncertain prenatal results, we argue in favour of the autonomy of women and their right to information from genome-wide CMA in order to make informed choices about their pregnancies. We propose that information material to a woman's decision-making process, including uncertain information, should not be withheld, and that it would be paternalistic for clinicians to try to take responsibility for women's decisions to terminate pregnancies. Non-directive pre-test and post-test genetic counselling is central to the delivery of these ethical objectives.

TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families.

BACKGROUND: The TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK) and metatropic dysplasia (MD) are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes. METHODS AND RESULTS: We critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement. CONCLUSIONS: Our data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.

No evidence of RET germline mutations in familial pituitary adenoma.

Pituitary adenomas are common in the general population. Although most of them are sporadic, some occur in a familial setting. In familial pituitary adenoma patients it is common that no germline defects are found after screening of aryl hydrocarbon receptor interacting protein (AIP) and other genes known to underlie the condition, suggesting the existence of yet unknown predisposition genes. Recently, the RET proto-oncogene was found to be a novel in vivo interaction partner of AIP in the pituitary gland. Here, we have screened patients from 16 AIP mutation negative (AIPmut-) pituitary adenoma families for RET germline mutations to assess whether RET could play a role in pituitary adenoma predisposition, similar to AIP. We found five novel germline RET changes: one in RET Exon 4 and the rest in noncoding regions of RET. Two changes, c.1560*G > A and -1285 G > A, were segregated in affected family members. We also analyzed the RET region with enhancer element locator (EEL) to identify RET regulatory elements, and to see whether the changes resided in these. None of the variants mapped to the regions predicted by EEL. Expression of RET was examined in ten AIPmut- and seven AIP mutation positive (AIPmut+) somatotropinomas by immunohistochemistry, with a trend showing reduced expression in the latter (P = 0.05). We conclude that the RET variants are presumably not related to pituitary adenoma predisposition, although reduced RET expression may play a role in AIP-related genesis of somatotropinomas.

Periventricular heterotopia in common microdeletion syndromes.

Periventricular heterotopia (PH) is a brain malformation characterised by heterotopic nodules of neurons lining the walls of the cerebral ventricles. Mutations in FLNA account for 20-24% of instances but a majority have no identifiable genetic aetiology. Often the co-occurrence of PH with a chromosomal anomaly is used to infer a new locus for a Mendelian form of PH. This study reports four PH patients with three different microdeletion syndromes, each characterised by high-resolution genomic microarray. In three patients the deletions at 1p36 and 22q11 are conventional in size, whilst a fourth child had a deletion at 7q11.23 that was larger in extent than is typically seen in Williams syndrome. Although some instances of PH associated with chromosomal deletions could be attributed to the unmasking of a recessive allele or be indicative of more prevalent subclinical migrational anomalies, the rarity of PH in these three microdeletion syndromes and the description of other non-recurrent chromosomal defects do suggest that PH may be a manifestation of multiple different forms of chromosomal imbalance. In many, but possibly not all, instances the co-occurrence of PH with a chromosomal deletion is not necessarily indicative of uncharacterised underlying monogenic loci for this particular neuronal migrational anomaly.

Development of a multiplex ligation-dependent probe amplification assay for diagnosis and estimation of the frequency of spinocerebellar ataxia type 15.

BACKGROUND: Spinocerebellar ataxia type 15 (SCA15) is a slowly progressive neurodegenerative disorder characterized by cerebellar ataxia. Mutation of the ITPR1 gene (inositol 1,4,5-triphosphate receptor, type 1) has been identified recently as the underlying cause, and in most cases the molecular defect is a multiexon deletion. To date, 5 different SCA15 families have been identified with ITPR1 gene deletion. METHODS: We have designed a synthetic, dual-color multiplex ligation-dependent probe amplification (MLPA) assay that measures copy number with high precision in selected exons across the entire length of ITPR1 and the proximal region of the neighboring gene, SUMF1 (sulfatase modifying factor 1). We screened 189 idiopathic ataxic patients with this MLPA assay. RESULTS: We identified ITPR1 deletion of exons 1-10 in the previously reported AUS1 family (4 members) and deletion of exons 1-38 in a new family (2 members). In addition to the multiexon deletions, apparent single-exon deletions identified in 2 other patients were subsequently shown to be due to single-nucleotide changes at the ligation sites. CONCLUSIONS: The frequency of ITPR1 deletions is 2.7% in known familial cases. This finding suggests that SCA15 is one of the "less common" SCAs. Although the deletions in the 5 families identified worldwide thus far have been of differing sizes, all share deletion of exons 1-10. This region may be important, both in terms of the underlying pathogenetic mechanism and as a pragmatic target for an accurate, robust, and cost-effective diagnostic analysis.

A4T mutation in the SOD1 gene causing familial amyotrophic lateral sclerosis.

We report the clinical and laboratory findings in the largest kindred so far recorded with familial amyotrophic lateral sclerosis due to an A4T mutation in the SOD1 gene. The age of onset ranged from 32 to 60 years, with a mean of 46 years. Weakness in the legs was the most frequent early symptom and there was a predominance of lower motor neuron signs. The mean time from onset of symptoms to death was 14 months. One man with onset at the age of 37 has shown a slowly developing form and is currently alive 76 months after diagnosis (October 2002), although severely affected. The A4T mutation, with one exception, was of similar severity to the A4V mutation.

Identification of a haplosufficient 3.6-Mb region in human chromosome 11q14.3-->q21.

Cytogenetic deletions are almost always associated with phenotypic abnormality and are very rarely transmitted. We have located a hitherto undescribed, familial deletion involving the region 11q14.3-->q21 in five individuals in a three-generation kindred. Four of the deletion carriers show no phenotypic abnormality; the other, who is the proband, was investigated for short stature and poor academic progress. In view of the apparent innocuous nature of this genetic imbalance, the deletion was investigated in detail to determine its size (3.6 Mb) and location with reference to molecular markers and genetic content. The deleted region is described by a contig of 37 BACS including the flanking regions, which we have assembled. Several possible contributory factors are considered, which might explain the lack of clinical significance of this large deletion. It is notable that there are few genes in this region and none have known functions. All most likely have copies elsewhere in the genome and a number of other hypothetical genes appear to be members of certain gene families, i.e. none is unique. Part of the region (1 Mb) is also duplicated at the pericentromeric region 11p11. Given the very low proportion of the genome occupied by single copy genes and their uneven distribution, regions such as this, which appear to be functionally haplosufficient, may be more common than hitherto recognised.

Profuse familial adenomatous polyposis with an adenomatous polyposis coli exon 3 mutation.

The attenuated form of familial adenomatous polyposis coli (AAPC) is associated with mutations in the adenomatous polyposis coli (APC) gene which cluster in the 5' region of the gene. It has been proposed that a 'genotype-phenotype boundary' exists at codons 159-163, and mutations that are 5' of this boundary will produce AAPC. Herein we document a three-generation family with an exon 3 mutation well to the 5' side of the proposed boundary, in which two affected individuals have had, in their 40s, a profuse form of familial adenomatous polyposis coli. We conclude that the codon 159-163 'boundary' is indicative rather than definitive. These two patients also had postoperative intra-abdominal adhesions, severely so in one.

High frequency hearing loss correlated with mutations in the GJB2 gene.

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.

Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency.

We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.