Trent Burgess

Pathogenic aberrations revealed exclusively by single nucleotide polymorphism (SNP) genotyping data in 5000 samples tested by molecular karyotyping

Background Several recent studies have demonstrated the use of single nucleotide polymorphism (SNP) arrays for the investigation of intellectual disability, developmental delay, autism or congenital abnormalities. In addition to LogR ‘copy number’ data, these arrays provide SNP genotyping data for gene level autozygosity mapping, estimating low levels of mosaicism, assessing long continuous stretches of homozygosity (LCSH), detection of uniparental disomy, and ‘autozygous’ regions. However, there remains little specific information on the clinical utility of this genotyping data. Methods Molecular karyotyping, using SNP array, was performed on 5000 clinical samples. Results Clinically significant ‘LogR neutral’ genotyping abnormalities were detected in 0.5% of cases. Among these were a single case of chimerism, 12 cases with low level chromosome mosaicism, and 11 cases with an LCSH associated with uniparental disomy. In addition, the genotyping data revealed several LCSH associated with clinically relevant ‘recessive type’ genetic defects. Conclusions These results demonstrate the utility of SNP genotyping data for detection of clinically significant abnormalities, including chimerism/mosaicism and recessive Mendelian disorders associated with autozygosity. The incidence of clinically significant low level mosaicism inferred from these cases suggests that this has hitherto been underestimated and chromosome mosaicism frequently occurs in the absence of indicative clinical features. The growing appreciation among clinicians and demand for SNP genotyping data poses significant challenges for the interpretation of LCSH, especially where there is no detailed phenotypic description to direct laboratory analysis. Finally, reporting of unexpected or hidden consanguinity revealed by SNP array analysis raises potential ethical and legal issues.

Small CGG repeat expansion alleles of FMR1 gene are associated with parkinsonism

Fragile X‐associated tremor/ataxia syndrome (FXTAS) affects older males carrying premutation, that is, expansions of the CGG repeat (in the 55–200 range), in the FMR1 gene. The neurological changes are linked to the excessive FMR1 messenger RNA (mRNA), becoming toxic through a ‘gain‐of‐function’. Because elevated levels of this mRNA are also found in carriers of the smaller expansion (grey zone) alleles, ranging from 40 to 54 CGGs, we tested for a possible role of these alleles in the origin of movement disorders associated with tremor.

A model for offering carrier screening for fragile X syndrome to nonpregnant women: results from a pilot study.

Purpose: To develop a model of offering population carrier screening for fragile X syndrome to nonpregnant women in primary care, using a program evaluation framework.Methods: A three-phase approach included: (I) needs assessment exploring staff and client attitudes, and informing development of educational materials, questionnaires and protocols; (II) offering screening to women, with questionnaires at baseline (Q1) and another (Q2) 1-month later; (III) genetic counseling for test-positive women and interviews with a subgroup of participants.Results: Of 338 volunteering for Phase II, 94% completed Q1, 59% completed Q2, and 20% (N = 65) chose testing revealing one premutation carrier and three gray zone results; 31 women were interviewed. Tested women had more positive attitudes toward screening (Q1: P < 0.001; Q2: P < 0.001) compared with untested, although there was no significant difference in mean knowledge scores or anxiety. Women generally supported being offered prepregnancy screening; however, reasons against being tested included: not currently planning a family; perceiving benefits of screening as unimportant; and having to return for testing.Conclusion: This is the first prospective study exploring informed decision-making for fragile X syndrome carrier screening, using a thorough process of consultation, with no apparent harms identified. It provides a model for development of future genetic screening programs.

FOXP1 mutations cause intellectual disability and a recognizable phenotype

Mutations in FOXP1, located at 3p13, have been reported in patients with global developmental delay (GDD), intellectual disability (ID), and speech defects. Mutations in FOXP2, located at 7q31, are well known to cause developmental speech and language disorders, particularly developmental verbal dyspraxia (DVD). FOXP2 has been shown to work co‐operatively with FOXP1 in mouse development. An overlap in FOXP1 and FOXP2 expression, both in the songbird and human fetal brain, has suggested that FOXP1 may also have a role in speech and language disorders. We report on a male child with a 0.19 MB intragenic deletion that is predicted to result in haploinsufficiency of FOXP1. Review of our patient and others reported in the literature reveals an emerging phenotype of GDD/ID with moderate to severe speech delay where expressive speech is most severely affected. DVD appears not to be a distinct feature in this group. Facial features include a broad forehead, downslanting palpebral fissures, a short nose with broad tip, relative or true macrocephaly, a frontal hair upsweep and prominent digit pads. Autistic traits and other behavioral problems are likely to be associated with haploinsufficiency of FOXP1. Congenital malformations may be associated.

High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy.

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low‐resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation‐Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy‐number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty‐two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost‐effective, high‐throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.

4.45 Mb microduplication in chromosome band 14q12 including FOXG1 in a girl with refractory epilepsy and intellectual impairment.

Microdeletions at 14q12 that include FOXG1, or loss of function mutations in FOXG1, are associated with the congenital variant of Rett syndrome. By SNP microarray analysis we identified a corresponding microduplication at 14q12 in a nine year old girl with symptomatic generalised epilepsy, severe intellectual impairment, and minor dysmorphisms, but without microcephaly. The 14q12 microduplication comprised 4.45 Mb of DNA and included FOXG1. This is the first report of duplication involving FOXG1 and suggests a dosage sensitive role for FOXG1 in brain development.

An improved Diagnostic PCR Assay for identification of Cryptic Heterozygosity for CGG Triplet Repeat Alleles in the Fragile X Gene (FMR1)

BackgroundFragile X syndrome (OMIM #300624) is the most common, recognised, heritable cause of mental retardation. Widespread testing is warranted by the relatively high frequency of the disorder, the benefits of early detection and the identification of related carriers whose offspring are at a 1 in 2 risk of inheriting the expanded pathogenic mutation. However, cost-effective screening of mentally retarded individuals has been impeded by the lack of a single, simple laboratory test. Currently, Fragile X syndrome can be excluded in males and a majority of females using a simple high-throughput PCR test. Due to the limited sensitivity of the PCR test, we find in our diagnostic service that approximately 40% of females appear homozygous and a labour intensive and expensive Southern blot test is required to distinguish these from females carrying one normal allele and an expanded allele.ResultsWe describe an improved PCR test which displays a high level of precision allowing alleles differing by a single triplet to be resolved. Using the new assay, we detected 46/83 (53%) cryptic heterozygotes previously labelled as homozygotes. The assay also extended the range of repeats amplifiable, up to 170 CGG repeats in males and 130 CGG repeats in females. Combined with the high precision, the assay also improves discrimination of normal (CGG repeats < 45) from grey zone (45 < CGG repeats < 54) alleles and grey zone alleles from small premutations (55 < CGG repeats < 100).ConclusionUse of this PCR test provides significantly improved precision and amplification of longer alleles. The number of follow-up Southern blot tests required is reduced (up to 50%) with consequent improvement in turnaround time and cost.

Phenotypic variability of distal 22q11.2 copy number abnormalities

The availability of microarray technology has led to the recent recognition of copy number abnormalities of distal chromosome 22q11.2 that are distinct from the better‐characterized deletions and duplications of the proximal region. This report describes five unrelated individuals with copy number abnormalities affecting distal chromosome 22q11.2. We report on novel phenotypic features including diaphragmatic hernia and uterine didelphys associated with the distal microdeletion syndrome; and frontomedial polymicrogyria and callosal agenesis associated with the distal microduplication syndrome. We describe the third distal chromosome 22q11.2 microdeletion patient with Goldenhar syndrome. Patients with distal chromosome 22q11.2 copy number abnormalities exhibit inter‐ and intra‐familial phenotypic variability, and challenge our ability to draw meaningful genotype–phenotype correlations.

Extending the scope of diagnostic chromosome analysis: Detection of single gene defects using high-resolution SNP microarrays†

Microarray analysis has provided significant advances in the diagnosis of conditions resulting from submicroscopic chromosome abnormalities. It has been recommended that array testing should be a “first tier” test in the evaluation of individuals with intellectual disability, developmental delay, congenital anomalies, and autism. The availability of arrays with increasingly high probe coverage and resolution has increased the detection of decreasingly small copy number changes (CNCs) down to the intragenic or even exon level. Importantly, arrays that genotype SNPs also detect extended regions of homozygosity. We describe 14 examples of single gene disorders caused by intragenic changes from a consecutive set of 6,500 tests using high‐resolution SNP microarrays. These cases illustrate the increased scope of cytogenetic testing beyond dominant chromosome rearrangements that typically contain many genes. Nine of the cases confirmed the clinical diagnosis, that is, followed a “phenotype to genotype” approach. Five were diagnosed by the laboratory analysis in the absence of a specific clinical diagnosis, that is, followed a “genotype to phenotype” approach. Two were clinically significant, incidental findings. The importance of astute clinical assessment and laboratory‐clinician consultation is emphasized to optimize the value of microarrays in the diagnosis of disorders caused by single gene copy number and sequence mutations. 32:1500–1506, 2011. ©2011 Wiley Periodicals, Inc.

Psychological status in female carriers of premutation FMR1 allele showing a complex relationship with the size of CGG expansion.

We utilized a sample of 299 adult females aged between 19 and 86 years, carrying fragile X mental retardation (FMR1) alleles with small CCG expansions ranging from 50 to 141 repeats to analyse the relationships between psychological symptoms as assessed by the Symptom Checklist‐90‐Revised (SCL‐90‐R) and the size of the CGG repeat in the FMR1 gene. There were highly significant (negative) correlations between the size of the CGG repeat and a great majority of SCL‐90‐R subscale scores and all the global indices, suggesting that carriers of premutations in the mid‐size CGG repeat range may be at greatest risk for the development of psychiatric disorder.