Microarray testing in clinical diagnosis: an analysis of 5,300 New Zealand patients.

BACKGROUND: The use of Microarray (array CGH) analysis has become a widely accepted front-line test replacing G banded chromosome studies for patients with an unexplained phenotype. We detail our findings of over 5300 cases. RESULTS: Of 5369 pre and postnatal samples, copy number variants (CNVs) were detected in 28.3 %, of which ~40 % were deletions and ~60 % were duplications. 96.8 % of cases with a CNV <5 Mb would not have been detected by G banding. At least 4.9 % were determined to meet the minimum criteria for a known syndrome. Chromosome 17 provided the greatest proportion of pathogenic CNVs with 65 % classified as (likely) pathogenic. X chromosome CNVs were the most commonly detected accounting for 4.2 % of cases, 0.7 % of these being classified as cryptic (likely) pathogenic CNVs. CONCLUSIONS: Microarray analysis as a primary testing strategy has led to a significant increase in the detection of CNVs (~29 % overall), with ~9 % carrying pathogenic CNVs and one syndromic case identified per 20 referred patients. We suggest these frequencies are consistent with other heterogeneous studies. Conversely, (likely) pathogenic X chromosome CNVs appear to be greater compared with previous studies.

The Diagnosis of Choriocarcinoma in Molar Pregnancies: A Revised Approach in Clinical Testing.

BACKGROUND: Hydatidiform moles occur in approximately 1 in 1,500 pregnancies; however, early miscarriages or spontaneous abortions may not be correctly identified as molar pregnancies due to poor differentiation of chorionic villi. METHODS: The current clinical testing algorithm used for the detection of hydatidiform moles uses a combination of morphological analysis and p57 immunostaining followed by ploidy testing to establish a diagnosis of either a complete or partial molar pregnancy. We review here 198 referrals for fluorescence in situ hybridization (FISH) ploidy testing, where the initial diagnosis based on morphology is compared to the final diagnosis based on a combination of morphology, FISH and p57 immunohistochemical (IHC) staining. RESULTS: Approximately 40% of cases were determined to be genetically abnormal, but only 28.8% of cases were diagnosed as molar pregnancies. The underestimation of complete molar pregnancies and those with androgenetic inheritance was also found to be likely using conventional diagnostic methods, as atypical p57 staining was observed in approximately 10% of cases. CONCLUSIONS: Our findings suggest that a revised approach to testing products of conception is necessary, with cases screened according to their clinical history in order to distinguish molar pregnancy referrals from hydropic pregnancies.

12q14 Microdeletions: Additional Case Series with Confirmation of a Macrocephaly Region.

To date, there have been only a few reports of patients carrying a microdeletion in chromosome 12q14. These patients usually present with pre- and postnatal growth retardation, and developmental delay. Here we report on two additional patients with both genotype and phenotype differences. Similar to previously published cases, one patient has haploinsufficiency of the HMGA2 gene and shows severe short stature and developmental delay. The second patient is only one of a handful without the loss of the HMGA2 gene and shows a much better growth profile, but with absolute macrocephaly. This patient's deletion is unique and hence defines a likely macrocephaly locus that contributes to the general phenotype characterising the 12q14 syndrome.

SNP Analysis and Whole Exome Sequencing: Their Application in the Analysis of a Consanguineous Pedigree Segregating Ataxia.

Autosomal recessive cerebellar ataxia encompasses a large and heterogeneous group of neurodegenerative disorders. We employed single nucleotide polymorphism (SNP) analysis and whole exome sequencing to investigate a consanguineous Maori pedigree segregating ataxia. We identified a novel mutation in exon 10 of the SACS gene: c.7962T>G p.(Tyr2654*), establishing the diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). Our findings expand both the genetic and phenotypic spectrum of this rare disorder, and highlight the value of high-density SNP analysis and whole exome sequencing as powerful and cost-effective tools in the diagnosis of genetically heterogeneous disorders such as the hereditary ataxias.

A turner syndrome patient carrying a mosaic distal x chromosome marker.

A skin sample from a 17-year-old female was received for routine karyotyping with a set of clinical features including clonic seizures, cardiomyopathy, hepatic adenomas, and skeletal dysplasia. Conventional karyotyping revealed a mosaic Turner syndrome karyotype with a cell line containing a small marker of X chromosome origin. This was later confirmed on peripheral blood cultures by conventional G-banding, fluorescence in situ hybridisation and microarray analysis. Similar Turner mosaic marker chromosome cases have been previously reported in the literature, with a variable phenotype ranging from the mild "classic" Turner syndrome to anencephaly, agenesis of the corpus callosum, complex heart malformation, and syndactyly of the fingers and toes. This case report has a phenotype that is largely discordant with previously published cases as it lies at the severe end of the Turner variant phenotype scale. The observed cytogenetic abnormalities in this study may represent a coincidental finding, but we cannot exclude the possibility that the marker has a nonfunctioning X chromosome inactivation locus, leading to functional disomy of those genes carried by the marker.

A case of 17q21.31 microduplication and 7q31.33 microdeletion, associated with developmental delay, microcephaly, and mild dysmorphic features.

Concurrent cryptic microdeletion and microduplication syndromes have recently started to reveal themselves with the advent of microarray technology. Analysis has shown that low-copy repeats (LCRs) have allowed chromosome regions throughout the genome to become hotspots for nonallelic homologous recombination to take place. Here, we report a case of a 7.5-year-old girl who manifests microcephaly, developmental delay, and mild dysmorphic features. Microarray analysis identified a microduplication in chromosome 17q21.31, which encompasses the CRHR1, MAPT, and KANSL1 genes, as well as a microdeletion in chromosome 7q31.33 that is localised within the GRM8 gene. To our knowledge this is one of only a few cases of 17q21.31 microduplication. The clinical phenotype of patients with this microduplication is milder than of those carrying the reciprocal microdeletions, and suggests that the lower incidence of the former compared to the latter may be due to underascertainment.

Microduplication of 3p26.3 implicated in cognitive development.

We report here a 34-month-old boy with global developmental delay referred for molecular karyotyping and fragile X studies. Molecular karyotype analysis revealed a microduplication in the 3p26.3 region involving part of the CHL1 and CNTN6 genes. Several deletions, one translocation, and one duplication have previously been described in this region of chromosome 3. The CHL1 gene has been proposed as a dosage-sensitive gene with a central role in cognitive development, and so the microduplication reported here appears to be implicated in our patient's phenotype.

Array comparative genomic hybridization identifies a heterozygous deletion of the entire KCNJ2 gene as a cause of sudden cardiac death.

BACKGROUND: Large gene rearrangements, not detectable by standard molecular genetic sequencing techniques, are present in a minority of patients with long QT syndrome. We aimed to screen for large rearrangements in genes responsible for long QT syndrome as part of the molecular autopsy of a 36-year-old woman who died suddenly and had a negative autopsy. A retrospective analysis of an ECG identified a long QT interval, but sequencing of known LQT genes was uninformative. METHODS AND RESULTS: Array comparative genomic hybridization was used to screen for deletions and duplications in 101 genes implicated in cardiac disorders and sudden death using a postmortem blood sample. A 542 kb deletion encompassing the entire KCNJ2 gene was identified in the decedent. The mother had electrocardiographic U-wave changes consistent with Andersen-Tawil syndrome and exaggerated by exercise but none of the characteristic noncardiac features. Fluorescence in situ hybridization confirmed the deletion in the decedent and established its presence in the mother. CONCLUSIONS: A novel application of array comparative genomic hybridization and fluorescence in situ hybridization has identified that long QT syndrome and sudden cardiac death may occur as a result of a deletion of an entire gene. The case also supports recent research suggesting that noncardiac features of Andersen-Tawil syndrome occur only with missense or minor gene rearrangements in the KCNJ2 gene, resulting in a dominant negative effect on Kir2.x channels.

Delineation of 2q32q35 deletion phenotypes: two apparent "proximal" and "distal" syndromes.

We report on three patients with interstitial deletions of the long arm of chromosome 2 involving bands 2q32.1-q35. They presented with wide-ranging phenotypic variation including facial dysmorphisms, cleft palate, learning difficulties, behavioural issues and severe heart defects. Microarray analysis confirmed an 8.6 Mb deletion in patients 1 and 2 and a 24.7 Mb deletion in patient 3. We discuss the genes involved in the deleted regions including MYO1B, GLS, FRZB, SATB2, and CPS1 and compare the phenotype with those reported in the literature. Taken together, these data suggest that there is a spectrum of disease severity such that patients with deletions encompassing the region of 2q32.1q32.2, which includes the FRZB gene, show an apparently milder phenotype compared to those that lie further distal in 2q32.3q35 that encompasses the SATB2 gene.

Implications of a Chr7q21.11 Microdeletion and the Role of the PCLO Gene in Developmental Delay.

We report here a 4-year-old boy with global developmental delay who was referred for karyotyping and fragile X studies. A small interstitial deletion on chromosome 7 at band 7q21 was detected in all cells examined. Subsequent molecular karyotype analysis gave the more detailed result of a 6.3 Mb heterozygous deletion involving the interstitial chromosome region 7q21.11. In this relatively gene-poor region, the presynaptic cytomatrix protein, Piccolo (PCLO) gene appears to be the most likely candidate for copy number loss leading to a clinical phenotype. G-banded chromosome analysis of the parents showed this deletion was inherited from the father. Molecular karyotype analysis of the father's genome confirmed that it was the same deletion as that seen in the son; however, the father did not share the severity of his son's phenotype. This cytogenetically-visible deletion may represent another example of a chromosomal rearrangement conferring a variable phenotype on different family members.

Bacterial artificial chromosomes (BACs)-on-Beads™ as a diagnostic platform for the rapid aneuploidy screening of products of conception.

The aim of the present study was to evaluate the use of KaryoLite™ bacterial artificial chromosomes (BACs)­on­Beads™ (BoBs) technology for the rapid screening of products of conception (POC). Validation and prospective studies were carried out on 85 and 95 patient samples, respectively. Validation studies had previously been analyzed using routine culture and G-banded karyotyping. BoBs resulted in an abnormality detection frequency of 27%, with a failure rate of <3%. The time required for processing was significantly lower compared with that of tissue culture. In conclusion, BoBs technology decreased the failure rate, while increasing the analytical sensitivity compared with G-banded karyotype analysis alone. Additionally, significant cost savings may be achieved with regard to the time of processing and analysis of specimens.

Clinical Outcomes and Counselling Issues regarding Partial Trisomy of Terminal Xp in a Child with Developmental Delay.

Female carriers of balanced translocations involving an X chromosome and an autosome offer genetic counselling challenges. This is in view of the number of possible meiotic outcomes, but also due to the impact of X chromosome-localised genes that are no longer subject to gene silencing through the X chromosome inactivation centre. We present a case where delineation of the extent of X chromosome-localised genes on the derivative autosome using molecular karyotyping offers critical information in the context of genetic counselling.

Array-based Identification of Copy Number Changes in a Diagnostic Setting: Simultaneous gene-focused and low resolution whole human genome analysis.

OBJECTIVES: The aim of this study was to develop and validate a comparative genomic hybridisation (CGH) array that would allow simultaneous targeted analysis of a panel of disease genes and low resolution whole genome analysis. METHODS: A bespoke Roche NimbleGen 12x135K CGH array (Roche NimbleGen Inc., Madison, Wisconsin, USA) was designed to interrogate the coding regions of 66 genes of interest, with additional widely-spaced backbone probes providing coverage across the whole genome. We analysed genomic deoxyribonucleic acid (DNA) from 20 patients with a range of previously characterised copy number changes and from 8 patients who had not previously undergone any form of dosage analysis. RESULTS: The custom-designed Roche NimbleGen CGH array was able to detect known copy number changes in all 20 patients. A molecular diagnosis was also made for one of the additional 4 patients with a clinical diagnosis that had not been confirmed by sequence analysis, and carrier testing for familial copy number variants was successfully completed for the remaining four patients. CONCLUSION: The custom-designed CGH array described here is ideally suited for use in a small diagnostic laboratory. The method is robust, accurate, and cost-effective, and offers an ideal alternative to more conventional targeted assays such as multiplex ligation-dependent probe amplification.

The p.Ala510Val mutation in the SPG7 (paraplegin) gene is the most common mutation causing adult onset neurogenetic disease in patients of British ancestry.

The c.1529C >T change in the SPG7 gene, encoding the mutant p.Ala510Val paraplegin protein, was first described as a polymorphism in 1998. This was based on its frequency of 3 % and 4 % in two separate surveys of controls in the United Kingdom (UK) population. Subsequently, it has been found to co-segregate with disease in a number of different populations. Yeast expression studies support its having a deleterious effect. In this paper a consanguineous sibship is described in which four members who are homozygous for the p.Ala510Val variant present with a spectrum of disease. This spectrum encompasses moderately severe hereditary spastic paraparesis (HSP) with more minor ataxia in two siblings, moderately severe ataxia without spasticity in the third, and a very mild gait ataxia in the fourth. Two of the siblings also manifest vestibular failure. The remaining eight unaffected siblings are either heterozygous for the p.Ala510Val variant, or do not carry it at all. Homozygosity mapping using a high-density SNP array across the whole genome found just 11 genes (on two regions of chromosome 3) outside the SPG7 region on chromosome 16, which were homozygously shared by the affected siblings, but not shared by the unaffected siblings; none of them are likely to be causative. The weight of evidence is strongly in favour of the p.Ala510Val variant being a disease-causing mutation. We present additional data from the Auckland City Hospital neurogenetics clinic to show that the p.Ala510Val mutation is prevalent amongst HSP patients of UK extraction belying any suggestion that European p.Ala510Val haplotypes harbour a disease-causing mutation which the UK p.Ala510Val haplotypes do not. Taken together with previous findings of a carrier frequency of 3-4 % in the UK population (giving a homozygosity rate of 20-40/100,000), the data imply that the p.Ala510Val is the most common mutation causing neurogenetic disease in adults of UK ancestry, albeit the penetrance may be low or the disease caused may be mild.

Developmental delay referrals and the roles of Fragile X testing and molecular karyotyping: a New Zealand perspective.

Global developmental delay (GDD) affects ~1-3% of children, many of whom will also have intellectual disability (ID). Fragile X is the major genetic cause of GDD with mental retardation (MR) in males, accounting for ~20% of all X-linked MR. As Fragile X has serious genetic implications, the overwhelming majority of developmental delay (DD) cases referred to our laboratory are concerned with the exclusion of a diagnosis of Fragile X, along with simultaneous karyotype analysis to confirm chromosome aberrations. Critically, molecular laboratories have generally experienced a falling positive detection frequency of Fragile X. In this context, the recent implementation of array­based techno-logy has significantly increased the likelihood of detecting chromosome aberrations that underpin DD. In the current study, we report a Fragile X mutation detection frequency for DD referrals that is comparable with the falling UK detection frequencies. In addition, we find that there is a 9­fold greater likelihood of detecting clinically significant chromosomal aberrations than of detecting a full Fragile X mental retardation 1 (FMR1) gene CGG repeat expansion in cases referred on the basis of DD. We propose a more efficent sequential testing algorithm that involves an initial molecular karyotype, cascading to FMR1 gene analysis in the event of a negative result.

A novel microdeletion syndrome at 9q21.13 characterised by mental retardation, speech delay, epilepsy and characteristic facial features.

The increased use of array-CGH and SNP-arrays for genetic diagnosis has led to the identification of new microdeletion/microduplication syndromes and enabled genotype-phenotype correlations to be made. In this study, nine patients with 9q21 deletions were investigated and compared with four previously Decipher reported patients. Genotype-phenotype comparisons of 13 patients revealed several common major characteristics including significant developmental delay, epilepsy, neuro-behavioural disorders and recognizable facial features including hypertelorism, feature-less philtrum, and a thin upper lip. The molecular investigation identified deletions with different breakpoints and of variable lengths, but the 750 kb smallest overlapping deleted region includes four genes. Among these genes, RORB is a strong candidate for a neurological phenotype. To our knowledge, this is the first published report of 9q21 microdeletions and our observations strongly suggest that these deletions are responsible for a new genetic syndrome characterised by mental retardation with speech delay, epilepsy, autistic behaviour and moderate facial dysmorphy.

Amino-Terminal Microdeletion within the CNTNAP2 Gene Associated with Variable Expressivity of Speech Delay.

The contactin-associated protein-like 2 (CNTNAP2) gene is highly expressed in the frontal lobe circuits in the developing human brain. Mutations in this gene have been associated with several neurodevelopmental disorders such as autism and specific language impairment. Here we describe a 450 kb deletion within the CNTNAP2 gene that is maternally inherited in two male siblings, but with a variable clinical phenotype. This variability is described in the context of a limited number of other cases reported in the literature. The in-frame intragenic deletion removes a critical domain of the CNTNAP2 protein, and this case also highlights the challenges of correlating genotype and phenotype.

A novel 2.3 mb microduplication of 9q34.3 inserted into 19q13.4 in a patient with learning disabilities.

Insertional translocations in which a duplicated region of one chromosome is inserted into another chromosome are very rare. We report a 16.5-year-old girl with a terminal duplication at 9q34.3 of paternal origin inserted into 19q13.4. Chromosomal analysis revealed the karyotype 46,XX,der(19)ins(19;9)(q13.4;q34.3q34.3)pat. Cytogenetic microarray analysis (CMA) identified a ~2.3Mb duplication of 9q34.3 → qter, which was confirmed by Fluorescence in situ hybridisation (FISH). The duplication at 9q34.3 is the smallest among the cases reported so far. The proband exhibits similar clinical features to those previously reported cases with larger duplication events.

A rare chromosome 3 imbalance and its clinical implications.

The duplication of chromosome 3q is a rare disorder with varying chromosomal breakpoints and consequently symptoms. Even rarer is the unbalanced outcome from a parental inv(3) resulting in duplicated 3q and a deletion of 3p. Molecular karyotyping should aid in precisely determining the length and breakpoints of the 3q+/3p- so as to better understand a child's future development and needs. We report a case of an infant male with a 57.5 Mb duplication from 3q23-qter. This patient also has an accompanying 1.7 Mb deletion of 3p26.3. The duplicated segment in this patient encompasses the known critical region of 3q26.3-q27, which is implicated in the previously reported 3q dup syndrome; however, the accompanying 3p26.3 deletion is smaller than the previously reported cases. The clinical phenotype of this patient relates to previously reported cases of 3q+ that may suggest that the accompanying 1.7 Mb heterozygous deletion is not clinically relevant. Taken together, our data has refined the location and extent of the chromosome 3 imbalance, which will aid in better understanding the molecular underpinning of the 3q syndrome.

Pure duplication of the distal long arm of chromosome 15 with ebstein anomaly and clavicular anomaly.

This report is of a patient with pure trisomy of 15q24-qter who presents with the rare Ebstein anomaly and a previously unreported skeletal anomaly. Chromosome microarray analysis allowed high-resolution identification of the extent of the trisomy and provided a means of achieving higher-resolution breakpoint data. The phenotypic expression of unbalanced chromosomal regions is a complex phenomenon, and fine mapping of the involved region, as described here, is only a first step on the path to its full understanding. Overexpression of the LINGO-1 and CSPG4 genes has been implicated in developmental delay seen in other patients with trisomy of 15q24-qter, but our patient is currently too young to ascertain developmental progress. The genetic underpinning of Ebstein anomaly and the skeletal anomaly reported here is unclear based on our high-resolution dosage mapping.