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Genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP). Analysis was restricted to predefined gene panels associated with the patient's phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnoses outside of the panel(s) applied. We propose a complementary method to rapidly identify pathogenic variants, including those missed by 100KGP methods.The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, loss-of-function variants in disease genes with a LOEUF score 0.2. We compared our findings with the corresponding patient's diagnostic reports.324/332 (98%) of the variants identified using DeNovoLOEUF were diagnostic or partially diagnostic (whereby the variant was responsible for some of the phenotype). We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients.We have demonstrated a highly specific and rapid method with a 98% positive predictive value that has good concordance with standard analysis, low false-positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered.

Ciliopathies may be classed as primary or motile depending on the underlying ciliary defect and are usually considered distinct clinical entities. Primary ciliopathies are associated with multisystem syndromes typically affecting the brain, kidney, and eye, as well as other organ systems such as the liver, skeleton, auditory system, and metabolism. Motile ciliopathies are a heterogenous group of disorders with defects in specialised motile ciliated tissues found within the lung, brain, and reproductive system, and are associated with primary ciliary dyskinesia, bronchiectasis, infertility and rarely hydrocephalus. Primary and motile cilia share defined core ultra-structures with an overlapping proteome, and human disease phenotypes can reflect both primary and motile ciliopathies. CEP164 encodes a centrosomal distal appendage protein vital for primary ciliogenesis. Human CEP164 mutations are typically described in patients with nephronophthisis-related primary ciliopathies but have also been implicated in motile ciliary dysfunction. Here we describe a patient with an atypical motile ciliopathy phenotype and biallelic CEP164 variants. This work provides further evidence that CEP164 mutations can contribute to both primary and motile ciliopathy syndromes, supporting their functional and clinical overlap, and informs the investigation and management of CEP164 ciliopathy patients.

Context Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood. Objective We investigated genetic causes of PAI in children and young people over a 25 year period. Design, Setting and Participants Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers. Intervention and Outcome Measurements Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018). Results A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1/steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause. Conclusions PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved.

Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24–60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31+/– human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.

Pathogenic variants within PAX6 are most often associated with aniridia, but have been linked with other phenotypes such as nystagmus, cataracts and foveal hypoplasia. Data are presented from a large cohort of 434 probands referred for PAX6 diagnostic testing. This analysis identified a wide range of pathogenic variants (n = 145) in 254 probands (including 61 novel variants). Excluding missense variants predicted to affect splicing, all 29 of the remaining missense variants were located within the paired (n = 27) or homeobox (n = 2) domains of the PAX6 protein, providing further evidence that these domains are critical to normal PAX6 function. Genotype-phenotype evidence suggests that while aniridia is associated with most variant types, a much broader clinical spectrum is seen in patients harbouring a missense variant, or a frameshift or run-on variant that results in an elongated or extended PAX6 protein.

Blepharophimosis, Ptosis, and Epicanthus inversus Syndrome (BPES) is caused by autosomal dominant mutations in FOXL2. There are two forms of BPES: type I (with primary ovarian insufficiency (POI)) and type II (without POI). Data are presented from a large cohort of 177 BPES probands. Diagnostic testing identified a wide range of mutations in 119 mutation-positive patients (including 38 novel mutations). Although FOXL2 mutations are distributed throughout the gene, over 50% were frameshift mutations within a hotspot region of the gene that can be detected using a single primer pair to provide a cost-effective and rapid screening method. There was a significant proportion of de novo cases in this study, although in 7% there may be undetected parental mosaicism. There was an excess of female compared to male probands and a highly significant bias in the parental original of inherited mutations, with 20/21 found to be paternal in origin (95%). This could be because BPES in a female is more likely to come to clinical attention and because there is a generalised and more widespread clinical effect on fertility, in addition to the established association with POI. This study demonstrates the importance of cascade screening and provides new information on inheritance and parental mosaicism in BPES which will aid genetic counselling and accurate risk management.

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories.Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software.Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity.Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics.

X chromosome inactivation (XCI) is an epigenetic mechanism that silences the majority of genes on one X chromosome in females. Previous studies have suggested that the spread of XCI might be facilitated in part by common repeats such as long interspersed nuclear elements (LINEs). However, owing to the unusual sequence content of the X and the nonrandom distribution of genes that escape XCI, it has been unclear whether the correlation between repeat elements and XCI is a functional one. To test the hypothesis that the spread of XCI shows sequence specificity, we have analyzed the pattern of XCI in autosomal chromatin by performing DNA methylation profiling in six unbalanced X;autosome translocations. Using promoter hypermethylation as an epigenetic signature of XCI, we have determined the inactivation status of 1050 autosomal genes after translocation onto an inactive derivative X. By performing a comparative sequence analysis of autosomal genes that are either subject to or escape the X inactivation signal, we identified a number of common repetitive elements, including L1 and L2 LINEs, and DNA motifs that are significantly enriched around inactive autosomal genes. We show that these same motifs predominantly map to L1P repeat elements, are significantly enriched on the X chromosome versus the autosomes and also occur at higher densities around X-linked genes that are subject to X inactivation compared with those that escape X inactivation. These results are consistent with a potential causal relationship between DNA sequence features such as L1s and the spread of XCI, lending strong support to Mary Lyon's 'repeat hypothesis'.

Leri–Weill dyschondrosteosis (LWD) results from heterozygous mutations of the SHOX gene, with homozygosity or compound heterozygosity resulting in the more severe form, Langer mesomelic dysplasia (LMD). These mutations typically take the form of whole or partial gene deletions, point mutations within the coding sequence, or large (>100?kb) 3? deletions of downstream regulatory elements. We have analyzed the coding sequence of the SHOX gene and its downstream regulatory regions in a cohort of 377 individuals referred with symptoms of LWD, LMD or short stature. A causative mutation was identified in 68% of the probands with LWD or LMD (91/134). In addition, a 47.5?kb deletion was found 160?kb downstream of the SHOX gene in 17 of the 377 patients (12% of the LWD referrals, 4.5% of all referrals). In 14 of these 17 patients, this was the only potentially causative abnormality detected (13 had symptoms consistent with LWD and one had short stature only), but the other three 47.5?kb deletions were found in patients with an additional causative SHOX mutation (with symptoms of LWD rather than LMD). Parental samples were available on 14/17 of these families, and analysis of these showed a more variable phenotype ranging from apparently unaffected to LWD. Breakpoint sequence analysis has shown that the 47.5?kb deletion is identical in all 17 patients, most likely due to an ancient founder mutation rather than recurrence. This deletion was not seen in 471 normal controls (P?0.0001), providing further evidence for a phenotypic effect, albeit one with variable penetration.

The 8p23.1 duplication syndrome is a relatively rare genomic condition that has been confirmed with molecular cytogenetic methods in only 11 probands and five family members. Here, we describe another prenatal and five postnatal patients with de novo 8p23.1 duplications analyzed with oligonucleotide array comparative genomic hybridization (oaCGH). Of the common features, mild or moderate developmental delays and/or learning difficulties have been found in 11/12 postnatal probands, a variable degree of mild dysmorphism in 8/12 and congenital heart disease (CHD) in 4/5 prenatal and 3/12 postnatal probands. Behavioral problems, cleft lip and/or palate, macrocephaly, and seizures were confirmed as additional features among the new patients, and novel features included neonatal respiratory distress, attention deficit hyperactivity disorder (ADHD), ocular anomalies, balance problems, hypotonia, and hydrocele. The core duplication of 3.68 Mb contains 31 genes and microRNAs of which only GATA4, TNKS, SOX7, and XKR6 are likely to be dosage sensitive genes and MIR124-1 and MIR598 have been implicated in neurocognitive phenotypes. A combination of the duplication of GATA4, SOX7, and related genes may account for the variable penetrance of CHD. Two of the duplications were maternal and intrachromosomal in origin with maternal heterozygosity for the common inversion between the repeats in 8p23.1. These additional patients and the absence of the 8p23.1 duplications in published controls, indicate that the 8p23.1 duplication syndrome may now be considered a pathogenic copy number variation (pCNV) with an estimated population prevalence of 1 in 58,000. © 2013 Wiley Periodicals, Inc.

Item does not contain fulltext Recent studies have identified PRDM9, a zinc finger (ZF) protein, as a key regulator of meiotic recombination. As both recurrent genomic disorders and chromosomal non-disjunction are known to be associated with specific unusual patterns of recombination, we hypothesized a possible link between PRDM9 ZF variation and susceptibility to microdeletion syndromes and/or trisomy. We sequenced the PRDM9 ZF domain in 271 parents of patients with de novo microdeletions of known parental origin (velocardiofacial syndrome, the 17q21.31 microdeletion syndrome, Prader-Willi/Angelman syndrome and Williams-Beuren syndrome), and in 61 parents of individuals with a supernumerary X chromosome. We compared PRDM9 ZF genotype frequencies between parents in whose germ line the de novo rearrangement occurred and their spouses. We observed a significantly increased frequency (p = 0.006) of PRDM9 variants in parents who transmitted de novo 7q11.23 deletions to their offspring. These data suggest that certain PRDM9 alleles may be associated with an increased susceptibility to recurrent 7q11.23 microdeletions that cause Williams-Beuren syndrome. However, as the majority of parents who transmitted a de novo microdeletion/supernumerary X chromosome to their offspring have the common AA genotype, we conclude that none of the rearrangements we have studied are dependent on specific non-A PRDM9 alleles.

We report a large series of 173 patients with physical and/or neurological abnormalities and a de novo imbalance identified by array CGH. Breakpoint intervals were screened for the presence of low copy repeats (LCRs) to distinguish between rearrangements formed by non-allelic homologous recombination (NAHR) and rearrangements formed by other mechanisms. We identified significant differences in size and parental origin between the LCR-mediated and non-LCR groups. Non-LCR imbalances were evenly distributed among the four size intervals we defined, whereas LCR-mediated rearrangements had a narrow size distribution, predominantly between 1 and 5 Mb (P=0.001). Among the LCR-mediated rearrangements there were equal numbers of maternally and paternally derived cases. In contrast, for the non-LCR rearrangements there was a significant excess of paternal cases (P=0.024) over a wide size range including below 1 Mb. Our results provide novel evidence that unbalanced chromosome rearrangements are not only more frequent in males, but may also arise through different mechanisms than those seen in females. Although the paternal imbalances identified in our study are evenly distributed throughout the four size groups, there are very few maternal imbalances either 10 Mb. Furthermore, a lower proportion of paternal imbalances are LCR mediated (13/71) compared with the maternal imbalances (12/30). We hypothesise that imbalances of maternal origin arise predominantly through NAHR during meiosis, while the majority of imbalances of paternal origin arise through male-specific mechanisms other than NAHR. Our data suggest that mitotic mechanisms could be important for the formation of chromosome imbalances; however, we found no association with increased paternal age.

We present an infant with a de novo cytogenetically visible interstitial deletion of approximately 21.9 Mb involving chromosome bands 7p15.1–7p12.1, with the loss of 119 genes confirmed by array CGH. The infant had a ventricular septal defect, hand and skull anomalies, and hyperglycaemia compatible with haploinsufficiency of TBX20, GLI3, and GCK genes, respectively. In addition, the infant had some features reminiscent of Beckwith Wiedemann syndrome including macroglossia, umbilical hernia, and a relatively large birth weight and we speculate that this is due to the deletion of GRB10, an imprinted gene on chromosome 7. This report illustrates how knowledge of genes within a deleted interval facilitates optimal medical management, can explain observed phenotypes, and stimulates research questions.

Background: Congenital chromosome abnormalities are relatively common in our species and among structural abnormalities the most common class is balanced reciprocal translocations. Determining the parental origin of de novo balanced translocations may provide insights into how and when they arise. While there is a general paternal bias in the origin of non-recurrent unbalanced rearrangements, there are few data on parental origin of non-recurrent balanced rearrangements. Methods: The parental origin of a series of de novo balanced reciprocal translocations was determined using DNA from flow sorted derivative chromosomes and linkage analysis. Results: Of 27 translocations, we found 26 to be of paternal origin and only one of maternal origin. We also found the paternally derived translocations to be associated with a significantly increased paternal age (p

Deletions of the SHOX gene are well documented and cause disproportionate short stature and variable skeletal abnormalities. In contrast interstitial SHOX duplications limited to PAR1 appear to be very rare and the clinical significance of the only case report in the literature is unclear. Mapping of this duplication has now shown that it includes the entire SHOX gene but little flanking sequence and so will not encompass any of the long-range enhancers required for SHOX transcription. We now describe the clinical and molecular characterization of three additional cases. The duplications all included the SHOX coding sequence but varied in the amount of flanking sequence involved. The probands were ascertained for a variety of reasons: hypotonia and features of Asperger syndrome, Leri-Weill dyschondrosteosis (LWD), and a family history of cleft palate. However, the presence of a duplication did not correlate with any of these features or with evidence of skeletal abnormality. Remarkably, the proband with LWD had inherited both a SHOX deletion and a duplication. The effect of the duplications on stature was variable: height appeared to be elevated in some carriers, particularly in those with the largest duplications, but was still within the normal range. SHOX duplications are likely to be under ascertained and more cases need to be identified and characterized in detail in order to accurately determine their phenotypic consequences.

The majority of constitutional reciprocal translocations appear to be unique rearrangements arising from independent events. However, a small number of translocations are recurrent, most significantly the t(11;22)(q23;q11). Among large series of translocations there may be multiple independently ascertained cases with the same cytogenetic breakpoints. Some of these could represent additional recurrent rearrangements, alternatively they could be identical by descent (IBD) or have subtly different breakpoints when examined under higher resolution. We have used molecular breakpoint mapping and haplotyping to determine the origin of three pairs of reciprocal constitutional translocations, each with the same cytogenetic breakpoints. FISH mapping showed one pair to have different breakpoints and thus to be distinct rearrangements. Another pair of translocations were IBD with identical breakpoint intervals and highly conserved haplotypes on the derived chromosomes. The third pair, t(4;11)(p16.2;p15.4), had the same breakpoint intervals by aCGH and fosmid mapping but had very different haplotypes, therefore they represent a novel recurrent translocation. Unlike the t(11;22)(q23;q11), the formation of the t(4;11)(p16.2;p15.4) may have involved segmental duplications and sequence homology at the breakpoints. Additional examples of recurrent translocations could be identified if the resources were available to study more translocations using the approaches described here. However, like the t(4;11)(p16.2;p15.4), such translocations are likely to be rare with the t(11;22) remaining the only common recurrent constitutional reciprocal translocation.

Duplications of distal 8p with and without significant clinical phenotypes have been reported and are often associated with an unusual degree of structural complexity. Here, we present a duplication of 8p23.1-8p23.2 ascertained in a child with speech delay and a diagnosis of ICD-10 autism. The same duplication was found in his mother who had epilepsy and learning problems. A combination of cytogenetic, FISH, microsatellite, MLPA and oaCGH analysis was used to show that the duplication extended over a minimum of 6.8 Mb between 3 539 893 and 10 323 426 bp. This interval contains 32 novel and 41 known genes, of which only microcephalin (MCPH1) is a plausible candidate gene for autism at present. The distal breakpoint of the duplicated region interrupts the CSMD1 gene in 8p23.2 and the medial breakpoint lies between the MSRA and RP1L1 genes in 8p23.1.An interchromosomal insertion between a normal and polymorphically inverted chromosome 8 is proposed to explain the origin of this duplication. Further mapped imbalances of distal 8p are needed to determine whether the autistic component of the phenotype in this family results from the cumulative imbalance of many genes or dosage imbalance of an individual susceptibility gene.

Donnai-Barrow syndrome [Faciooculoacousticorenal (FOAR) syndrome; DBS/FOAR] is a rare autosomal recessive disorder resulting from mutations in the LRP2 gene located on chromosome 2q31.1. We report a unique DBS/FOAR patient homozygous for a 4-bp LRP2 deletion secondary to paternal uniparental isodisomy for chromosome 2. The propositus inherited the mutation from his heterozygous carrier father, whereas the mother carried only wild-type LRP2 alleles. This is the first case of DBS/FOAR resulting from uniparental disomy (UPD) and the fourth published case of any paternal UPD 2 ascertained through unmasking of an autosomal recessive disorder. The absence of clinical symptoms above and beyond the classical phenotype in this and the other disorders suggests that paternal chromosome 2 is unlikely to contain imprinted genes notably affecting either growth or development. This report highlights the importance of parental genotyping in order to give accurate genetic counseling for autosomal recessive disorders.

We report the analyses of breakpoints in 31 phenotypically normal and 14 abnormal carriers of balanced translocations. Our study assesses the differences between balanced translocations in normal carriers and those in abnormal carriers, focusing on the presence of genomic imbalances at the breakpoints or elsewhere in the genome, presence of cryptic chromosome rearrangements, and gene disruption. Our hypothesis is that all four features will be associated with phenotypic abnormalities and absent or much less frequent in a normal population. In the normal cohort, we identified neither genomic imbalances at the breakpoints or elsewhere in the genome nor cryptic chromosome rearrangements. In contrast, we identified candidate disease-causing imbalances in 4/14 abnormal patients. These were three breakpoint associated deletions and three deletions unrelated to the breakpoints. All six de novo deletions originated on the paternally inherited chromosome. Additional complexity was also present in one of these cases. Gene disruption by the breakpoints was present in 16/31 phenotypically normal individuals and in 5/14 phenotypically abnormal patients. Our results show that translocations in phenotypically abnormal patients are molecularly distinct from those in normal individuals: the former are more likely to be associated with genomic imbalances at the breakpoints or elsewhere and with chromosomal complexity, whereas the frequency of gene disruption is similar in both normal and abnormal translocation carriers.

Human chromosome 2 contains large blocks of segmental duplications (SDs), both within and between proximal 2p and proximal 2q, and these may contribute to the frequency of the common variant inversion inv(2)(p11.2q13). Despite their being cytogenetically homogeneous, we have identified four different breakpoint combinations by fluorescence in situ hybridization mapping of 40 cases of inv(2)(p11.2q13) of European origin. For the vast majority of inversions (35/40), the breakpoints fell within the same spanning BACs, which hybridized to both 2p11.2 and 2q13 on the normal and inverted homologues. Sequence analysis revealed that these BACs contain a significant proportion of intrachromosomal SDs with sequence homology to the reciprocal breakpoint region. In contrast, BACs spanning the rare breakpoint combinations contain fewer SDs and with sequence homology only to the same chromosome arm. Using haplotype analysis, we identified a number of related family subgroups with identical or very closely related haplotypes. However, the majority of cases were not related, demonstrating for the first time that the inv(2)(p11.2q13) is a truly recurrent rearrangement. Therefore, there are three explanations to account for the frequent observation of the inv(2)(p11.2q13): the majority have arisen independently in different ancestors, while a minority either have been transmitted from a common founder or have different breakpoints at the molecular cytogenetic level. Udgivelsesdato: 2007-Oct

The pericentric inv(10)(p11.2q21.2) mutation has been frequently identified in cytogenetic laboratories, is phenotypically silent, and is considered to be a polymorphic variant. Cloning and sequencing of the junction fragments on 10p11 and 10q21 revealed that neither inversion breakpoint directly involved any genes or repetitive sequences, although both breakpoint regions contain a number of repeats. All 20 apparently unrelated inv(10) families in our study had identical breakpoints, and detailed haplotype analysis showed that the inversions were identical by descent. Thus, although considered a common variant, inv(10)(p11.2q21.2) has a single ancestral founder among northern Europeans.

Non-allelic homologous recombination between chromosome-specific LCRs is the most common mechanism leading to recurrent microdeletions and duplications. To look for locus-specific differences, we have used microsatellites to determine the parental and chromosomal origins of a large series of patients with de novo deletions of chromosome 7q11.23 (Williams syndrome), 15q11-q13 (Angelman syndrome, Prader-Willi syndrome) and 22q11 (Di George syndrome) and duplications of 15q11-q13. Overall the majority of rearrangements were interchromosomal, so arising from unequal meiotic exchange, and there were approximately equal numbers of maternal and paternal deletions. Duplications and deletions of 15q11-q13 appear to be reciprocal products that arise by the same mechanisms. The proportion arising from interchromosomal exchanges varied among deletions with 22q11 the highest and 15q11-q13 the lowest. However, parental and chromosomal origins were not always independent. For 15q11-q13, maternal deletions tended to be interchromosomal while paternal deletions tended to be intrachromosomal; for 22q11 there was a possible excess of maternal cases among intrachromosomal deletions. Several factors are likely to be involved in the formation of recurrent rearrangements and the relative importance of these appear to be locus-specific.

Leri-Weill dyschondrosteosis (LWD) is a pseudoautosomal dominant disorder characterized by disproportionate short stature and a characteristic curving of the radius, known as the "Madelung deformity." SHOX mutations resulting in SHOX haploinsufficiency have been found in LWD and in a variable proportion of patients with idiopathic short stature (ISS), whereas homozygous loss of SHOX results in the more severe Langer mesomelic dysplasia (LMD). Defects in SHOX have been identified in approximately 60% of LWD cases, whereas, in the remaining approximately 40%, the molecular basis is unknown. This suggests either genetic heterogeneity or the presence of mutations in unanalyzed regions of SHOX, such as the upstream, intragenic, or downstream regulatory sequences. Therefore, the pseudoautosomal region 1 (PAR1) of 80 patients with LWD, in whom SHOX deletions and mutations had been excluded, was screened for deletions by use of a new panel of microsatellite markers. We identified 12 patients with LWD who presented with a novel class of PAR1 deletions that did not include SHOX. The deletions were of variable size and mapped at least approximately 30-530 kb downstream of SHOX. In our cohort, this type of deletion accounted for 15% of cases. In all cases, the deletions cosegregated with the phenotype. No apparent phenotypic differences were observed between patients with SHOX deletions and those with this new class of PAR1 deletions. Thus, we present here the identification of a second PAR1 region implicated in the etiopathogenesis of LWD. Our findings suggest the presence of distal regulatory elements of SHOX transcription in PAR1 or, alternatively, the existence of an additional locus apparently involved in the control of skeletal development. Deletion analysis of this newly identified region should be included in the mutation screening of patients with LWD, LMD, and ISS.

Objectives: The frequency of abnormalities of 15q11-q13 and other possibly causal medical disorders including karyotypic abnormalities was investigated in an unselected series of children who were referred to one of two autism assessment centres. Methods: Two hundred and twenty-one cases were assessed using the Autism Diagnostic Interview and Observation Schedule and, where appropriate, standardized tests of intelligence and language abilities. Medical histories and notes were reviewed, and molecular and cytogenetic investigations used to detect chromosomal abnormalities. Results: One hundred and eighty-one cases were diagnosed according to International Classification of Diseases - version 10 criteria as having an autism spectrum disorder (autistic-like Pervasive Developmental Disorder) and 40 cases as having other disorders. Twenty-one (11.6%) of the children with autism spectrum disorders had a possibly causal condition compared with six (15%) of the children with other diagnoses. One child with an autism spectrum disorder had a paternally inherited familial duplication of 15q11-13. The pattern of genotype-phenotype correlation within the family indicated that this form of abnormality might carry a risk for developmental difficulties, although the risk did not appear to be specific for autism spectrum disorders. Conclusion: The overall rate of possibly causal medical and cytogenetic conditions in children with autism spectrum disorders was low and no different from the rate of disorder in children with other developmental/neuropsychiatric disorders that attended the same clinics. Further research is required to determine whether paternal duplication of 15q11-13 gives rise to adverse developmental outcomes.

Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes.

Duplications involving the X chromosome, in which the duplicated region is not subject to inactivation, are rare. We describe four distal Xq duplications, in three males and one female, in which the duplicated X chromosomal material is active in all cells. The infantile phenotype bears some resemblance to that of the Prader-Willi syndrome, presenting with initial feeding difficulties, hypotonia and, sometimes, with cryptorchidism. However, the severity of the phenotype is not simply related to the size of the duplication and so variations in gene expression, gene disruption or position effects from breakpoints should be considered as explanations. We have compared the clinical, cytogenetic and molecular findings of our patients with those previously reported. This has enabled us to question the suggestion that duplication of the gene SOX3 is the cause of hypopituitarism and that duplication of Filamin A is the cause of bilateral periventricular nodular heterotopia/mental retardation syndrome (BPNH/MR). We have also narrowed the putative critical interval for X-linked spina bifida.

The amygdala, which plays a critical role in emotional learning and social cognition, is structurally and functionally sexually dimorphic in humans. We used magnetic neuroimaging and molecular genetic analyses with healthy subjects and patients possessing X-chromosome anomalies to find dosage-sensitive genes that might influence amygdala development. If such X-linked genes lacked a homologue on the Y-chromosome they would be expressed in one copy in normal 46,XY males and two copies in normal 46,XX females. We showed by means of magnetic neuroimaging that 46,XY males possess significantly increased amygdala volumes relative to normal 46,XX females. However, females with Turner syndrome (45,X) have even larger amygdalae than 46,XY males. This finding implies that haploinsufficiency for one or more X-linked genes influences amygdala development irrespective of a direct or indirect (endocrinological) mechanism involving the Y-chromosome. 45,X females also have increased grey matter volume in the orbitofrontal cortex bilaterally, close to a region implicated in emotional learning. They are as poor as patients with bilateral amygdalectomies in the recognition of fear from facial expressions. We attempted to localize the gene(s) responsible for these deficits in X-monosomy by means of a deletion mapping strategy. We studied female patients possessing structural X-anomalies of the short arm. A genetic locus (no greater than 4.96 Mb in size) at Xp11.3 appears to play a key role in amygdala and orbitofrontal structural and (by implication) functional development. Females with partial X-chromosome deletions, in whom this critical locus is deleted, have normal intelligence. Their fear recognition is as poor as that of 45,X females and their amygdalae are correspondingly enlarged. This 4.96 Mb region contains, among others, the genes for monoamine oxidase A (MAOA) and B (MAOB), which are involved in the oxidative deamination of several neurotransmitters, including dopamine and serotonin. Abnormal activity of these neurotransmitters has been implicated in the aetiology of several neurodevelopmental disorders in which social cognitive deficits are prominent. These associated deficits include a specific lack of fear recognition from facial expressions. We show that the thrombocytic activity of MAOB is proportionate to the number of X-chromosomes, and hypothesize that haploinsufficiency of this enzyme in 45,X females predisposes to their deficits in social cognition.

Sequence comparison analysis has been carried out for 31 imprinted mouse genes and a set of 150 control genes. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable elements (SINEs), in particular SINE Alu repeats. This is similar to recent analyses of human imprinted genes and supports the suggestion that there is either active selection against SINE elements in imprinted regions or a reduced rate of insertion of these elements. The reduction in numbers of SINEs was more consistent in paternally expressed genes, whereas for maternally expressed genes significantly reduced numbers of SINE-B2 elements were coupled with increased numbers of SINE-B4 and SINE-ID elements. Paternally expressed genes were also found to be associated with a lower GC content. Discriminant analysis revealed that the two sub-groups of imprinted genes can be cleanly separated from each other on the basis of their genomic sequence characteristics and that they tend to localize to different genomic compartments. The differences between the sequence characteristics of imprinted and control genes have also enabled us to develop a discriminant function that can be used in a genome-wide screen to identify candidate imprinted genes.

Translocations occur in a proportion of couples affected by recurrent miscarriages. We describe two such families in which the underlying cause was a cryptic subtelomeric 11p;17p translocation detected only after the birth of an affected child carrying an unbalanced form of the rearrangement. Unbalanced subtelomeric rearrangements are now recognised as a significant cause of mental impairment and we believe that these rearrangements may also be an important cause of recurrent miscarriages. In these two families the translocation is most likely to have arisen from a single ancestral event because all translocation carriers shared almost identical haplotypes around the breakpoints on both chromosomes.

Human trisomy is attributable to many different mechanisms and the relative importance of each mechanism is highly chromosome specific. The association between altered recombination and maternal non-disjunction is well documented: reductions in recombination have been reported for maternal meiosis I (MI) errors involving chromosomes 15, 16, 18 and 21 and increased recombination has been reported for meiosis II (MII) errors involving chromosome 21. We therefore investigated maternal X chromosome non-disjunction, to determine whether the effects of recombination are unique to the X chromosome or similar to any of the autosomes thus far studied. We genotyped 45 47,XXX females and 95 47,XXY males of maternal origin. Our results demonstrate that 49% arose during MI, 29% during MII and 16% were postzygotic events; a further 7% were meiotic but could not be assigned as either MI or MII because of recombination at the centromere. Among the MI cases, a majority (56%) had no detectable transitions and so absent recombination is an important factor for X chromosome non-disjunction. However, similar to trisomy 15 and unlike trisomy 21, we observed a significant increase in the mean maternal age of transitional MI errors compared with nullitransitional cases. In our studies of MII errors, recombination appeared normal and there was no obvious effect of maternal age, distinguishing our results from MII non-disjunction of chromosomes 18 or 21. Thus, surprisingly, the risk factors associated with both MI and MII non-disjunction appear to be different for virtually every chromosome that has been adequately studied.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

Integrity of the long arm of the X chromosome is important for maintaining female fertility and several critical regions for normal ovarian function have been proposed. In order to understand further the importance of specific areas of the X chromosome, we describe a series of 20 previously unreported patients missing part of Xq in whom detailed phenotypic information has been gathered as well as precise chromosome mapping using array Comparative Genomic Hybridization. Features often associated with Turner syndrome were not common in our study and excluding puberty, menarche and menstruation, the phenotypes observed were present in only a minority of women and were not specific to the X chromosome. The most frequently occurring phenotypic features in our patients were abnormalities of menstruation and fertility. Larger terminal deletions were associated with a higher incidence of primary ovarian failure, occurring at a younger age; however patients with similar or even identical deletions had discordant menstrual phenotypes, making accurate genetic counselling difficult. Nevertheless, large deletions are likely to be associated with complete skewing of X inactivation so that the resulting phenotypes are relatively benign given the amount of genetic material missing, even in cases with unbalanced X;autosome translocations. Some degree of ovarian dysfunction is highly likely, especially for terminal deletions extending proximal to Xq27. In conjunction with patient data from the literature, our study suggests that loss of Xq26-Xq28 has the most significant effect on ovarian function.

Only a small number of individuals with duplications within the proximal short arm of the X chromosome have been reported. The majority of patients have duplications encompassing Xp11-p21, or extend more distally into Xp22. We report on a female patient who presented within the first year of life with plagiocephaly, speech delay, and epilepsy. Brain MRI showed a relatively thin cerebral cortex, abnormal periventricular white matter, and abnormal vessels in the left inferior parietal region. Cytogenetic and microsatellite analysis of the patient and her parents showed that she has a de novo duplication of Xp11.22-Xp11.4 on her paternal X chromosome. FISH analysis using fluorescently labeled BACs followed by array analysis including an X tilepath BAC array showed that a 12.3 Mb interval between 40.4 Mb and 52.7 Mb from the Xp telomere (NCBI build 36) was duplicated and excluded the presence of additional rearrangements along the X chromosome. Interestingly, X-inactivation studies in peripheral blood leukocytes showed that the duplicated (paternal) X chromosome was active in the majority of cells, in contrast to other patients with Xp duplications in whom X inactivation is random or skewed toward the normal X. These findings suggest that overexpression of genes from proximal Xp is likely to have contributed to her clinical phenotype.

A significant proportion of both pericentric and paracentric inversions have recurrent breakpoints and so could either have arisen through multiple independent events or be identical by descent (IBD) with a single common ancestor. Of two common variant inversions previously studied, the inv(2)(p11q13) was genuinely recurrent while the inv(10)(p11.2q21.2) was IBD in all cases tested. Excluding these two variants we have ascertained 257 autosomal inversion probands at the Wessex Regional Genetics Laboratory. There were 104 apparently recurrent inversions, representing 35 different breakpoint combinations and we speculated that at least some of these had arisen on more than one occasion. However, haplotype analysis identified no recurrent cases among eight inversions tested, including the variant inv(5)(p13q13). The cases not IBD were shown to have different breakpoints at the molecular cytogenetic level. No crossing over was detected within any of the inversions and the founder haplotypes extended for variable distances beyond the inversion breakpoints. Defining breakpoint intervals by FISH mapping identified no obvious predisposing elements in the DNA sequence. In summary the vast majority of human inversions arise as unique events. Even apparently recurrent inversions, with the exception of the inv(2)(p12q13), are likely to be either derived from a common ancestor or to have subtly different breakpoints. Presumably the lack of selection against most inversions allows them to accumulate and disperse amongst different populations over time.

Zellweger syndrome (ZS) is an autosomal recessive peroxisomal disorder that results from mutations in one of the peroxisome biogenesis (PEX) genes. This is the first patient reported with uniparental disomy (UPD) resulting in ZS, in this case maternal isodisomy of chromosome 1 involving reduction to homoallelism of a frameshift mutation within PEX 10. Other reported cases of UPD1, and evidence for the imprinting of genes on chromosome 1, are reviewed. The molecular findings in this patient have important implications for molecular testing and genetic counseling in ZS.

We report the parental origin, and where possible the chromosomal origin of 115 de novo unbalanced structural chromosome abnormalities detectable by light microscopy. These consisted of 39 terminal deletions, 35 interstitial deletions, 8 rings, 12 duplications and 21 unbalanced translocations. In all categories the majority of abnormalities were of paternal origin, although the proportions varied from a high of 84% in the interstitial deletions and rings to a low of 58% in the duplications. Among the interstitial deletions and duplications, there were approximately equal numbers of intra- and interchromosomal abnormalities, while the majority of unbalanced translocations were isodisomic for the duplicated chromosome. The examination of the parental ages in the four main classes of abnormality showed terminal deletions of maternal origin to be associated with a significantly reduced maternal age. Thus, there is a clear propensity for structural chromosome abnormalities to occur in male germ cells, although the chromosomal origin seems similar irrespective of the parental origin.

Background: Studies of chromosome 15 abnormality have implicated over-expression of paternally imprinted genes in the 15q11–13 region in the aetiology of autism. To test this hypothesis we compared individuals with Prader-Willi syndrome (PWS) due to uniparental disomy (UPD – where paternally imprinted genes are over-expressed) to individuals with the 15q11–13 deletion form of the syndrome (where paternally imprinted genes are not over-expressed). We also tested reports that PWS cases due to the larger type I (TI) form of deletion show differences to cases with the smaller type II (TII) deletion. Method: Ninety-six individuals with PWS were recruited from genetic centres and the PWS association. Forty-nine individuals were confirmed as having maternal UPD of chromosome 15 and were age and sex matched to 47 individuals with a deletion involving 15q11–13 (32 had the shorter (T II) deletion, and 14 had the longer (TI) deletion). Behavioural assessments were carried out blind to genetic status, using the Autism Diagnostic Observation Schedule (ADOS), the Autism Diagnostic Interview (ADI), the Autism Screening Questionnaire (ASQ), the Children's Yale-Brown Obsessive-Compulsive Scale (CY-BOCS), the Vineland Adaptive Behaviour Scales (VABS), and measurements of intellectual ability, including the Wechsler and Mullen Scales and Raven's Matrices. Results: UPD cases exhibited significantly more autistic-like impairments in reciprocal social interaction on questionnaire, interview and standardised observational measures. Comparison of TI and TII deletion cases revealed few differences, but ability levels tended to be lower in the TI deletion cases. Conclusions: Findings from a large study comparing deletion and UPD forms of Prader-Willi syndrome were consistent with other evidence in indicating that paternally imprinted genes in the 15q11–13 region constitute a genetic risk factor for aspects of autistic symptomatology. These genes may therefore play a role in the aetiology of autism. By contrast with another report, there was no clear-cut relationship between the size of the deletion and the form of cognitive and behavioural phenotype.

Segmental aneuploidy usually has phenotypic consequences but unbalanced rearrangements without phenotypic consequences have also been reported. In particular, harmless deletions of G-dark bands 5p14 and 16q21 have each been found in more than one independent family. Here, we report two families that were ascertained at prenatal diagnosis and had similar overlapping deletions that removed most of the gene poor G-dark band 2p12. PCR mapping showed that the deletions had a minimum size of 6.1 and 6.9 Mb with at least 13 hemizygous loci including a cluster of six pancreatic islet-regenerating genes. These deletions had no apparent phenotypic consequences in eight family members. In contrast, a third family was ascertained through a child with Wilm's tumour; both the child and his mother had more proximal deletions, developmental delay and some dysmorphic features. The deletion had a minimum size of 5.7 Mb and extended into the gene-rich area of 2p11.2. These results are consistent with the idea that there may be segments of the genome that are consistently haplosufficient. The introduction of higher resolution methods of dosage analysis into diagnostic laboratories is already revealing more transmitted abnormalities of uncertain significance. As a result, published cases of transmitted imbalances have been collected as a guide to the possible significance of such findings in the future (see the 'Chromosome Anomaly Collection' at www.som.soton.ac.uk/research/geneticsdiv).