Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

Partial trisomy 2p and partial monosomy 2q arising from a paternal intrachromosomal 2q-into-2p between-arm insertion and paracentric inversion: molecular cytogenetic characterization of a four-break rearrangement.

We report on a 26-month-old boy with an interstitial duplication of 2p22.3p22.2 and an interstitial deletion of 2q14.1q21.2. The abnormality was derived from his father having a balanced paracentric inversion and pericentric insertion. The deletion in the child was identified by cytogenetic analysis and characterized in more detail by molecular cytogenetics and array comparative genomic hybridization. The latter revealed a 20-Mb deletion in the long arm and a 5.6-Mb duplication in the short arm of chromosome 2. Fluorescence in situ hybridization in paternal chromosomes characterized an intrachromosomal insertion of 2q14.1q21.2 into 2p23; additionally a paracentric inversion of 2p13p23 was observed. The boy with the unbalanced karyotype suffered from severe psychomotor retardation, thrombophilia due to protein C deficiency, and hypertrophic cardiomyopathy and also had phenotypic abnormalities. Most of these features have previously been described in individuals with interstitial deletion of 2q14.1.

Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching.

Cross-species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty-six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the "text string' option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification.

Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes.

We have localized the DNA sequences required for mitotic centromere function on the human Y chromosome. Analysis of 33 rearranged Y chromosomes allowed the centromere to be placed in interval 8 of a 24-interval deletion map. Although this interval is polymorphic in size, it can be as small as approximately 500kb. It contains alphoid satellite DNA and approximately 300kb of adjacent Yp sequences. Chromosomes with rearrangements in this region were analysed in detail. Two translocation chromosomes and one monocentric isochromosome had breakpoints within the alphoid array. Of 12 suppressed Y centromeres on translocation chromosomes and dicentric isochromosomes that were also analysed two showed deletions one of which only removed alphoid DNA. These results indicate that alphoid DNA is a functional part of the Y chromosome centromere.

Identification of de novo mutations and rare variants in hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is one of the most severe congenital heart malformations, characterized by underdevelopment of the structures in the left heart-aorta complex. The majority of cases are sporadic. Although multiple genetic loci have been tentatively implicated in HLHS, no gene or pathway seems to be specifically associated with the disease. To elucidate the genetic basis of HLHS, we analyzed 53 well-characterized patients with isolated HLHS using an integrated genomic approach that combined DNA sequencing of five candidate genes (NKX2-5, NOTCH1, HAND1, FOXC2 and FOXL1) and genome-wide screening by high-resolution array comparative genomic hybridization. In 30 patients, we identified two novel de novo mutations in NOTCH1, 23 rare patients inherited gene variants in NOTCH1, FOXC2 and FOXL1, and 33 rare patients mostly inherited copy-number variants. Some of the identified variations coexisted in the same patient. The biological significance of such rare variations is unknown, but our findings strengthen the role of NOTCH pathway in cardiac valve development, indicating that HLHS is, at least in part, a 'valve' disease. This is the first report of de novo mutations associated with isolated HLHS. Moreover, the coexistence of multiple rare variants suggests in some cases a cumulative effect, as shown for other complex disease.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Refining the phenotype associated with MEF2C haploinsufficiency.

Recently, submicroscopic deletions of the 5q14.3 region have been described in patients with severe mental retardation (MR), stereotypic movements, epilepsy and cerebral malformations. Further delineation of a critical region of overlap in these patients pointed to MEF2C as the responsible gene. This finding was further reinforced by the identification of a nonsense mutation in a patient with a similar phenotype. In brain, MEF2C is essential for early neurogenesis, neuronal migration and differentiation. Here we present two additional patients with severe MR, autism spectrum disorder and epilepsy, carrying a very small deletion encompassing the MEF2C gene. This finding strengthens the role of this gene in severe MR, and enables further delineation of the clinical phenotype.

Eyebrow anomalies as a diagnostic sign of genomic disorders.

Microdeletions and microduplications in the human genome, termed genomic disorders, contribute to a high proportion of human multisystemic neurodevelopmental diseases and are detected by array-based comparative genomic hybridization (aCGH). In general, most genomic disorders are associated with craniofacial and skeletal features and behavioural abnormalities, in addition to learning disability and developmental delay (LD/DD). Specifically, recognition of a characteristic 'facial gestalt' has been the key to distinguish one genomic disorder from the other. Here, we report our experience concerning the relevance of abnormal eyebrow pattern as a diagnostic indicator of specific genomic disorders.

Epigenetic analysis of the critical region I for premature ovarian failure: demonstration of a highly heterochromatic domain on the long arm of the mammalian X chromosome.

BACKGROUND: X chromosome rearrangements defined a critical region for premature ovarian failure (POF) that extended for >15 Mb in Xq. It has been shown previously that the region could be divided into two functionally distinct portions and suggested that balanced translocations interrupting its proximal part, critical region 1 (CR1), could be responsible for POF through downregulation of ovary expressed autosomal genes translocated to the X chromosome. RESULTS AND CONCLUSION: This study reports that such position effect can indeed be demonstrated by analysis of breakpoint regions in somatic cells of POF patients and by the finding that CR1 has a highly heterochromatic organisation, very different from that of the euchromatic autosomal regions involved in the rearrangements. The chromatin organisation of the POF CR1 is likely to be responsible for the epigenetic modifications observed in POF patients. The characteristics of CR1 and its downregulation in oocytes may very well explain its role in POF and the frequency of the POF phenotype in chromosomal rearrangements involving Xq. This study also demonstrates a large and evolutionary conserved domain of the long arm of the X chromosome, largely corresponding to CR1, that may have structural or functional roles, in oocyte maturation or in X chromosome inactivation.

Chromosomal microarray mapping suggests a role for BSX and Neurogranin in neurocognitive and behavioral defects in the 11q terminal deletion disorder (Jacobsen syndrome).

We performed a prospective analysis on 14 11q- patients to determine the relationship between the degree of cognitive impairment and relative deletion size. Seventeen measures of cognitive function were assessed. All nine patients with a deletion of at least 12.1 Mb had severe global cognitive impairment, with full-scale IQ <50, whereas all five patients with smaller deletions,

Inverted duplications deletions: underdiagnosed rearrangements??

Molecular techniques led to the discovery that several chromosome rearrangements interpreted as terminal duplications were in fact inverted duplications contiguous to terminal deletions. Inv dup del rearrangements originate through a symmetric dicentric chromosome that, after asymmetric breakage, generates an inv dup del and a deleted chromosome. In recurrent inverted duplications the dicentric chromosome is formed at meiosis through non-allelic homologous recombination. In non-recurrent inv dup del cases, dicentric intermediates are formed by non-homologous end joining or intrastrand annealing. Some authors hypothesized that in these cases the dicentric may have been formed directly in the zygote. Healing of the broken dicentric chromosomes can occur not only in a telomerase-dependent way but also through telomere capture and circularization thus creating translocated or ring inv dup del chromosomes. In all the cases reported up to now, the duplicated region was always longer than the deleted one, but we can safely assume that there is another group of rearrangements where the deleted region is longer than the duplicated portion. In general, in these cases, the cytogeneticist will suspect the presence of a deletion and confirm it by FISH with a subtelomeric probe, but he/she will almost certainly miss the duplication. It is likely that the conventional analysis techniques used until now have led to a substantial underestimate of the frequency of inv dup del rearrangements and that the widespread use of array-CGH in routine analysis will allow a more realistic estimate. Obviously, the concomitant presence of deletion and duplication has important consequences in genotype/phenotype correlations.

Duplications in addition to terminal deletions are present in a proportion of ring chromosomes: clues to the mechanisms of formation.

BACKGROUND AND METHODS: Ring chromosomes are often associated with abnormal phenotypes because of loss of genomic material at one or both ends. In some cases no deletion has been detected and the abnormal phenotype has been attributed to mitotic ring instability. We investigated 33 different ring chromosomes in patients with phenotypic abnormalities by array based comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). RESULTS: In seven cases we found not only the expected terminal deletion but also a contiguous duplication. FISH analysis in some of these cases demonstrated that the duplication was inverted. Thus these ring chromosomes derived through a classical inv dup del rearrangement consisting of a deletion and an inverted duplication. DISCUSSION: Inv dup del rearrangements have been reported for several chromosomes, but hardly ever in ring chromosomes. Our findings highlight a new mechanism for the formation of some ring chromosomes and show that inv dup del rearrangements may be stabilised not only through telomere healing and telomere capture but also through circularisation. This type of mechanism must be kept in mind when evaluating possible genotype-phenotype correlations in ring chromosomes since in these cases: (1) the deletion may be larger or smaller than first estimated based on the size of the ring, with a different impact on the phenotype; and (2) the associated duplication will in general cause further phenotypic anomalies and might confuse the genotype-phenotype correlation. Moreover, these findings explain some phenotypic peculiarities which previously were attributed to a wide phenotypic variation or hidden mosaicism related to the instability of the ring.

Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis.

BACKGROUND: Patients with a microscopically visible deletion of the distal part of the long arm of chromosome 1 have a recognisable phenotype, including mental retardation, microcephaly, growth retardation, a distinct facial appearance and various midline defects including corpus callosum abnormalities, cardiac, gastro-oesophageal and urogenital defects, as well as various central nervous system anomalies. Patients with a submicroscopic, subtelomeric 1qter deletion have a similar phenotype, suggesting that the main phenotype of these patients is caused by haploinsufficiency of genes in this region. OBJECTIVE: To describe the clinical presentation of 13 new patients with a submicroscopic deletion of 1q43q44, of which nine were interstitial, and to report on the molecular characterisation of the deletion size. RESULTS AND CONCLUSIONS: The clinical presentation of these patients has clear similarities with previously reported cases with a terminal 1q deletion. Corpus callosum abnormalities were present in 10 of our patients. The AKT3 gene has been reported as an important candidate gene causing this abnormality. However, through detailed molecular analysis of the deletion sizes in our patient cohort, we were able to delineate the critical region for corpus callosum abnormalities to a 360 kb genomic segment which contains four possible candidate genes, but excluding the AKT3 gene.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome.

BACKGROUND: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. AIM: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. RESULTS: We estimate the prevalence of the syndrome to be 1 in 16,000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p<10(-5)). CONCLUSION: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

ZPLD1 gene is disrupted in a patient with balanced translocation that exhibits cerebral cavernous malformations.

The past few years have seen rapid advances in our understanding of the genetics and molecular biology of cerebral cavernous malformations (CCM) with the identification of the CCM1, CCM2, and CCM3 genes. Recently, we have recruited a patient with an X/3 balanced translocation that exhibits CCM. By fluorescent in situ hybridization analysis, sequence analysis tools and database mining procedures, we refined the critical region to an interval of 200-kb and identified the interrupted ZPLD1 gene. We detected that the mRNA expression level of ZPLD1 gene is consistently decreased 2.5-fold versus control (P=0.0006) with allelic loss of gene expression suggesting that this protein may be part of the complex signaling pathway implicated in CCM formation.

Cryptic deletions are a common finding in "balanced" reciprocal and complex chromosome rearrangements: a study of 59 patients.

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as "balanced" by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a "chromosomal phenotype" and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of "balanced" CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customized platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Prematurity, ventricular septal defect and dysmorphisms are independent predictors of pathogenic copy number variants: a retrospective study on array-CGH results and phenotypical features of 293 children with neurodevelopmental disorders and/or multiple congenital anomalies.

BACKGROUND: Since 2010, array-CGH (aCGH) has been the first-tier test in the diagnostic approach of children with neurodevelopmental disorders (NDD) or multiple congenital anomalies (MCA) of unknown origin. Its broad application led to the detection of numerous variants of uncertain clinical significance (VOUS). How to appropriately interpret aCGH results represents a challenge for the clinician. METHOD: We present a retrospective study on 293 patients with age range 1 month - 29 years (median 7 years) with NDD and/or MCA and/or dysmorphisms, investigated through aCGH between 2005 and 2016. The aim of the study was to analyze clinical and molecular cytogenetic data in order to identify what elements could be useful to interpret unknown or poorly described aberrations. Comparison of phenotype and cytogenetic characteristics through univariate analysis and multivariate logistic regression was performed. RESULTS: Copy number variations (CNVs) with a frequency < 1% were detected in 225 patients of the total sample, while 68 patients presented only variants with higher frequency (heterozygous deletions or amplification) and were considered to have negative aCGH. Proved pathogenic CNVs were detected in 70 patients (20.6%). Delayed psychomotor development, intellectual disability, intrauterine growth retardation (IUGR), prematurity, congenital heart disease, cerebral malformations and dysmorphisms correlated to reported pathogenic CNVs. Prematurity, ventricular septal defect and dysmorphisms remained significant predictors of pathogenic CNVs in the multivariate logistic model whereas abnormal EEG and limb dysmorphisms were mainly detected in the group with likely pathogenic VOUS. A flow-chart regarding the care for patients with NDD and/or MCA and/or dysmorphisms and the interpretation of aCGH has been made on the basis of the data inferred from this study and literature. CONCLUSION: Our work contributes to make the investigative process of CNVs more informative and suggests possible directions in aCGH interpretation and phenotype correlation.

A large analphoid invdup(3)(q22.3qter) marker chromosome characterized by array-CGH in a child with malformations, mental retardation, ambiguous genitalia and Blaschko's lines.

We report a new case of mosaic chromosome 3-derived marker chromosome, present in fibroblasts but not in lymphocytes, found in a child with malformations, mental retardation and ambiguous genitalia. Cytogenetic and molecular analysis showed that the supernumerary invdup(3)(q22.3qter) chromosome was negative at FISH with alpha satellite probe. The presence of a functional neocentromere was confirmed by immunofluorescence with antibodies to centromere proteins (CENPs). Definition of the marker breakpoints has been done through array-CGH. The skin of the patient presented dyschromic areas ordered along Blaschko's lines. The invdup(3q) marker chromosome was present only in fibroblasts from the dark skin biopsy, while lymphocytes and fibroblasts from the normal skin showed a normal male karyotype. Expression of the HPS3 gene (MIM: 606118) was more than two times higher in dark skin fibroblasts. Neocentromeres are most often observed on chromosomal arm fragments that have separated from an endogenous centromere, and therefore actually confer mitotic stability to what would have been acentric fragments. To our knowledge, this invdup(3q) analphoid marker is the largest among the several reported so far. Parental origin and possible mode of formation have been defined by DNA polymorphisms studies. The size of the duplicated marker chromosome and its frequency and tissue distribution may be relevant to the severity of the propositus' phenotype.

2q24-q31 deletion: report of a case and review of the literature.

We report a patient with a de novo interstitial deletion of the long arm of chromosome 2 involving bands 2q24.3-q31.1. The patient shows postnatal growth retardation, microcephaly, ptosis, down-slanting palpebral fissures, long eyelashes and micrognathia. Halluces are long, broad and medially deviated, while the other toes are laterally deviated and remarkably short with hypoplastic phalanges. She also showed developmental delay, seizures, lack of eye contact, stereotypic and repetitive hand movements and sleep disturbances with breath holding. Prenatal and three independent postnatal karyotypes were normal. Array-CGH analysis allowed us to identify and characterize a "de novo" 2q interstitial deletion of about 10.4Mb, involving segment between cytogenetic bands 2q24.3 and 2q31.1. The deletion was confirmed by quantitative PCR. About 30 children with 2q interstitial deletion have been reported. The deletion described here is overlapping with 15 of these cases. We have attempted to compare the clinical features of our patient with 15 overlapping cases. The emerging phenotypes include low birth weight, postnatal growth retardation, mental retardation and developmental delay, microcephaly, and peculiar facial dysmorphisms. Peculiar long and broad halluces with an increased distance between the first and the second toe are ("sandal gap" sign) present in most of the described patients. The gene content analysis of the deleted region revealed the presence of some genes that may be indicated as good candidates in generating both neurological and dysmorphic phenotype in the patient. In particular, a cluster of SCNA genes is located within the deleted region and it is known that loss of function mutations in SCNA1 gene cause a severe form of epilepsy.

Identification of a recurrent breakpoint within the SHANK3 gene in the 22q13.3 deletion syndrome.

INTRODUCTION: The 22q13.3 deletion syndrome (MIM 606232) is characterised by neonatal hypotonia, normal to accelerated growth, absent to severely delayed speech, global developmental delay, and minor dysmorphic facial features. We report the molecular characterisation of the deletion breakpoint in two unrelated chromosome 22q13.3 deletion cases. METHODS: The deletions were characterised by FISH, checked for other abnormalities by array-CGH, and confirmed by Real-Time PCR, and finally the breakpoints were cloned, sequenced, and compared. RESULTS: Both cases show the cardinal features of the 22q13.3 deletion syndrome associated with a deletion involving the last 100 kb of chromosome 22q13.3. The cases show a breakpoint within the same 15 bp repeat unit, overlapping the results obtained by Wong and colleagues in 1997 and suggesting that a recurrent deletion breakpoint exists within the SHANK3 gene. The direct repeat involved in these 22q13 deletion cases is presumably able to form slipped (hairpin) structures, but it also has a strong potential for forming tetraplex structures. DISCUSSION: Three cases with a common breakpoint within SHANK3 share a number of common phenotypic features, such as mental retardation and developmental delay with severely delayed or absent expressive speech. The two cases presented here, having a deletion partially overlapping the commercial subtelomeric probe, highlight the difficulties in interpreting FISH results and suggest that many similar cases may be overlooked.