Joanne Whittaker

ALPHA -2 MACROGLOBULIN POLYMORPHISM AND ALZHEIMER DISEASE RISK IN THE UK

There is a genetic component in the aetiology of Alzheimer disease1 (AD). Recently, Blacker et al. reported an odds ratio of 3.56 for being affected by AD in individuals carrying at least one copy of an -2 macroglobulin gene (A2M) allele (A2M*2) corresponding to a deletion near the 5´ splice site of exon 18 (2). Cases were aged 50 years and older and 22% had autopsy diagnoses2. This effect was independent of APOE. We tried to replicate these findings using three different samples. Our first sample was composed of 125 autopsy-confirmed AD cases from Brain Banks in England and 218 elderly non-demented controls. Where age of onset was documented, 3 cases were younger than 50 years and 30 cases were 50-65 years. In cases where the age at onset was not available, 35 individuals died before age 66, including 2 before 50 years. We used non-demented elderly English individuals as controls: 59 from Ely (a town 14 miles from Cambridge) aged 70 years and older meeting neither DSM-III-R nor AGECAT dementia criteria3; and 159 from Cambridge aged 84 years and older with Mini Mental State Examination (MMSE) scores of more than 23 (3). Our second sample was composed of 53 pairs of age-matched (within 5 years) demented cases and controls, aged 70 years and older, from the epidemiologically based Ely component of the Medical Research Council Cognitive Function and Ageing Study4. Cases met both DSM-III-R and AGECAT criteria3 and controls met neither of these dementia criteria. The controls used in our first sample included those from the matched case-control series with six unmatched controls from the same cohort.

Identification of a mutation that perturbs NF1 agene splicing using genomic DNA samples and a minigene assay

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disease. In recent studies on the neurofibromatosis type 1 (NF1) gene neurofibromin, splicing abnormalities were seen in 30-50% of cases when RNA taken from cell lines was analysed.1,2 Unlike mutations that alter critical amino acids or generate premature stop codons, splicing abnormalities can be very hard to predict from sequence analysis alone. Apart from the two base pairs 5′ and 3′ of each exon, few of the nucleotides in regions critical for splicing are absolutely conserved. As a consequence, it can be very difficult to conclude that a sequence variation found in a patient will alter splicing and so represents a pathogenic mutation. ### Key points This difficulty is well illustrated by a family with NF1 in which we recently identified a sequence variation. The three generation family is from the UK and meets NIH diagnostic criteria. The index case, at the age of 82, has classical features of NF1 including multiple cafe au lait macules, neurofibromas, and axillary and inguinal freckling. Her son was similarly affected and died …

A Presenilin-1 Truncating Mutation Is Present in Two Cases with Autopsy-Confirmed Early-Onset Alzheimer Disease

We have examined genomic DNA from 40 cases of autopsy-confirmed early-onset Alzheimer disease (EOAD) (age at onset <=65 years) that were all unselected for family history. We have sequenced the 10 exons and flanking intronic sequences of the presenilin-1 (PS-1) gene for all 40 individuals. A single mutation, a deletion of a G from the intron 4 splice-donor consensus sequence, was detected in two individuals in this study. The mutation was associated with two shortened transcripts, both with shifted reading frames resulting in premature-termination codons. All the PS-1 mutations described elsewhere have been missense or in-frame splice mutations, and recent data suggest that these result in disease by gain-of-function or dominant-negative mechanisms. The mutation that we have identified is likely to result in haploinsufficiency and would be most consistent with other mutations acting in a dominant-negative manner. However, we cannot exclude the possibility that the small amounts of truncated transcripts exert a gain of function. Since no other mutations or polymorphisms were detected in our patients, mutations in the coding regions and splice consensus sequences of PS-1 are likely to be rare in EOAD cases unselected for family history.

Genotype-phenotype analysis in X-linked Emery–Dreifuss muscular dystrophy and identification of a missense mutation associated with a milder phenotype

Direct sequencing of the emerin gene in 22 families with Emery-Dreifuss muscular dystrophy (EMD) revealed mutations in 21 (95%), confirming that emerin mutations can be identified in the majority of families with X-linked EMD. Most emerin mutations result in absence of the protein. In this study three mutations (a missense mutation Pro183Thr and two in-frame deletions removing residues 95-99 and 236-241, respectively) were unusual in being associated with expression of mutant protein. The phenotype in these families was compared in detail with the clinical features in cases with typical null mutations. For the in-frame deletions there were no significant differences. In the family with the missense mutation the phenotype was milder. Age at onset was later for first symptoms and for development of ankle contractures and muscle weakness. These findings have diagnostic implications as well as pointing to functionally important regions of the emerin protein.

Automated comparative sequence analysis identifies mutations in 89% of NF1 patients and confirms a mutation cluster in exons 11–17 distinct from the GAP related domain

Neurofibromatosis type 1 (NF1), formerly known as Von Recklinghausen Neurofibromatosis, is a common genetic disorder affecting approximately 1 in 3000–5000 people. It is a fully penetrant autosomal dominant disorder. Strict diagnostic criteria that include cafe au lait spots, neurofibromas, plexiform neurofibromas, freckling in the axillary or inguinal regions, Lisch nodules (iris haematomas), optic or chiasma glioma, pseudoarthrosis, and sphenoid dysplasia define NF1. Most disease features are present in more than 90% of patients at puberty.1 Further manifestations are known to occur in this disorder, including macrocephaly, short stature, learning difficulties, scoliosis and certain malignancies.2–4 There is, however, great intra and interfamilial phenotypic variability. In addition a number of patients who have a clinical picture suspected to be NF1 do not fulfil the diagnostic criteria particularly in the younger age groups. As a consequence genetic testing would have a major impact on the diagnosis and management of these families. The NF1 gene maps to chromosome 17q11.2 and is thought to be a tumour suppressor gene because loss of heterozygosity is associated with the occurrence of benign and malignant tumours in tissues derived from the neural crest5–7 as well as myeloid malignancies.8 It spans a region of about 350 kb of genomic DNA and contains 60 exons.9–11 It harbours at least three other embedded genes—EV12A, EV12B, and ONGP—transcribed from the opposite strand of NF1 intron 27b. The NF1 gene transcribes several mRNAs in the size range 11–13 kb expressed in neurones, oligodendrocytes and non-myelinating Schwann cells.12 The most common transcript codes for a polypeptide of 2818 amino acids called neurofibromin.13–15 A 360 amino acid region of the predicted protein product shows homology with the GTPase activating (GAP) family of proteins in yeast and mammals.16 The GAP related domain (GRD) is …

Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS)

The association of the Noonan phenotype with neurofibromatosis type 1 (NF1) was first noted by Allanson et al. [Am J Med Genet 1985;21:457–462.] and 30 further cases have subsequently been reported. It has been suggested that this phenotype is more common than previously appreciated, as Colley et al. [Clin Genet 1996;49:59–64.] examined 94 sequentially identified patients with NF1 from their genetic register and found Noonan features in 12. A 3‐bp deletion of exon 17 of the NF1 neurofibromin gene was described in one family by Carey et al. [Proc Greenwood Genet Center 1997;17:52–53]. However, it remains unclear whether Neurofibromatosis–Noonan syndrome (NFNS) represents a form of NF1 (with mutations in the NF1 neurofibromin gene) or a separate syndrome. We have used a new, rapid sequence analysis technique—comparative sequence analysis (CSA)—to examine the NF1 gene in six patients with NFNS. None of the six patients had the previously identified mutation, nor did we observe other mutations within this exon. However, two other mutations were found: in exon 25, a 3‐bp deletion 4312 del GAA, and in exon 23‐2, a 2‐bp insertion 4095 ins TG. The PTPN11 gene, now known to cause over 50% of Noonan syndrome was also examined in four cases of NFNS, and no mutations were found. These results show that NFNS can in some cases result from different mutations in the NF1 gene and therefore represents a variant form of NF1.

Psychiatric symptoms and CAG repeats in neurologically asymptomatic Huntington's disease gene carriers

The putative relationship between the psychiatric profile of a sample of neurologically asymptomatic Huntingtons disease gene carriers and CAG repeats was investigated. The psychiatric assessments (by consultant psychiatrist and computerised battery) were undertaken before the genetic testing was carried out. In this way, the informational distortions caused by neurological and cognitive deficits were avoided. The hypothesis that there is a relationship between psychiatric and CAG repeats was tested by seeking direct correlations between psychiatric systems and CAG repeats, and also by correcting the correlation by the number of years above or below the estimated age of onset in Huntingtons disease. Scores for irritability and cognitive failures were high in the sample. There was no correlation between any psychiatric variable and CAG repeats. Possible explanations for this lack of correlations are discussed.

Presenilin-1 intron 8 polymorphism is not associated with autopsy-confirmed late-onset Alzheimer's disease.

Mutations in the presenilin-1 (PS-1) gene may account for the majority of familial early-onset Alzheimers disease (EOAD) cases. However, there is controversy as to whether the bi-allelic intron 8 PS-1 polymorphism plays a role in late-onset AD (LOAD). As previous association studies with this polymorphism have all investigated clinically diagnosed LOAD cases, we have analysed the frequency of the PS-1 intronic polymorphism in a series of autopsy-confirmed early- (n = 54) and late-onset (n = 199) cases and a large control population of non-demented, aged individuals (n = 215). Our sample size should have had the power to reveal effects of the size previously reported for the PS-1 polymorphism, but we detected no significant increase in the 1/1 risk genotype distribution in EOAD or LOAD cases. Thus, we have been unable to find an association between the PS-1 intronic polymorphism and early- or late-onset AD within this autopsy-confirmed population.

Comparative sequence analysis (CSA): A new sequence-based method for the identification and characterization of mutations in DNA

Direct sequencing analysis is largely used to confirm and characterize mutations previously detected by more rapid tests. We have developed a method—Comparative Sequence Analysis (CSA)—that simplifies the analysis of sequencing data facilitating its use as a first screen for mutation detection. Sequence data were split into their component electrophoretograms and the use of a size standard enabled equivalent traces from different individuals to be overlaid. This allowed simple and rapid visual analysis of the results. Using this technique in a blind study, we tested 576 samples for mutations in the Von Hippel‐Lindau tumor suppresser gene, VHL. We were able to identify and characterize all 78 known mutations present within the sample set (100% sensitivity and specificity). Hum Mutat 16:437–443, 2000.

Splicing analysis of unclassified variants in COL2A1 and COL11A1 identifies deep intronic pathogenic mutations

UK NHS diagnostic service sequence analysis of genes generally examines and reports on variations within a designated region 5′ and 3′ of each exon, typically 30 bp up and downstream. However, because of the degenerate nature of the splice sites, intronic variants outside the AG and GT dinucleotides of the acceptor and donor splice sites (ASS and DSS) are most often classified as being of unknown clinical significance, unless there is some functional evidence of their pathogenicity. It is now becoming clear that mutations deep within introns can also interfere with normal processing of pre-mRNA and result in pathogenic effects on the mature transcript. In diagnostic laboratories, these deep intronic variants most often fall outside of the regions analysed and so are rarely reported. With the likelihood that next generation sequencing will identify more of these unclassified variants, it will become important to perform additional studies to determine the pathogenicity of such sequence anomalies. Here, we analyse variants detected in either COL2A1 or COL11A1 in patients with Stickler syndrome. These have been analysed both in silico and functionally using either RNA isolated from the patients cells or, more commonly, minigenes as splicing reporters. We show that deep intronic mutations are not a rare occurrence, including one variant that results in multiple transcripts, where both de novo donor and ASS are created by the mutation. Another variant produces transcripts that result in either haploinsufficiency or a dominant negative effect, potentially modifying the disease phenotype.