Coexisting Conditions Modifying Phenotypes of Patients with 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder with an extremely broad phenotypic spectrum. The aim of our study was to investigate how often the additional variants in the genome can affect clinical variation among patients with the recurrent deletion. To examine the presence of additional variants affecting the phenotype, we performed microarray in 82 prenatal and 77 postnatal cases and performed exome sequencing in 86 postnatal patients with 22q11.2DS. Within those 159 patients where array was performed, 5 pathogenic and 5 likely pathogenic CNVs were identified outside of the 22q11.2 region. This indicates that in 6.3% cases, additional CNVs most likely contribute to the clinical presentation. Additionally, exome sequencing in 86 patients revealed 3 pathogenic (3.49%) and 5 likely pathogenic (5.81%) SNVs and small CNV. These results show that the extension of diagnostics with genome-wide methods can reveal other clinically relevant changes in patients with 22q11 deletion syndrome.

Comparative Genomic Hybridization to Microarrays in Fetuses with High-Risk Prenatal Indications: Polish Experience with 7400 Pregnancies.

The aim of this study was to determine the suitability of the comparative genomic hybridization to microarray (aCGH) technique for prenatal diagnosis, but also to assess the frequency of chromosomal aberrations that may lead to fetal malformations but are not included in the diagnostic report. We present the results of the aCGH in a cohort of 7400 prenatal cases, indicated for invasive testing due to ultrasound abnormalities, high-risk for serum screening, thickened nuchal translucency, family history of genetic abnormalities or congenital abnormalities, and advanced maternal age (AMA). The overall chromosomal aberration detection rate was 27.2% (2010/7400), including 71.2% (1431/2010) of numerical aberrations and 28.8% (579/2010) of structural aberrations. Additionally, the detection rate of clinically significant copy number variants (CNVs) was 6.8% (505/7400) and 0.7% (57/7400) for variants of unknown clinical significance. The detection rate of clinically significant submicroscopic CNVs was 7.9% (334/4204) for fetuses with structural anomalies, 5.4% (18/336) in AMA, 3.1% (22/713) in the group of abnormal serum screening and 6.1% (131/2147) in other indications. Using the aCGH method, it was possible to assess the frequency of pathogenic chromosomal aberrations, of likely pathogenic and of uncertain clinical significance, in the groups of cases with different indications for an invasive test.

Application of array comparative genomic hybridization (aCGH) for identification of chromosomal aberrations in the recurrent pregnancy loss.

Spontaneous abortion occurs in 8-20% of recognized pregnancies and usually takes place in the first trimester (7-11 weeks). There are many causes of pregnancy loss, but the most important (about 75%) is the presence of chromosomal aberrations. We present the results of oligonucleotide array application in a cohort of 62 miscarriage cases. The inclusion criteria for the study were the loss after 8th week of pregnancy and the appearance of recurrent miscarriages. DNA was extracted from trophoblast or fetal skin fibroblasts. In the 62 tested materials from recurrent miscarriages, the detection rate was 56.5% (35/62). The most commonly found were aneuploidies (65%) (chromosomal trisomy 14, 16, 18, 21, and 22), Turner syndrome, and triploidy (17.1%). Other chromosomal abnormalities included pathogenic and likely pathogenic structural aberrations: 1) pathogenic: deletion 7p22.3p12.3 and duplication 9p24.3p13.2 inherited from the normal father, deletion 3q13.31q22.2 and deletion 3q22.3q23 of unknown inheritance and duplication of 17p12 inherited from father with foot malformation; 2) likely pathogenic variants: deletion 17p13.1 inherited from normal mother, deletion 5q14.3 of unknown inheritance and de novo deletion 1q21.1q21.2. Among these aberrations, six CNVs (copy number variants) were responsible for the miscarriage: deletion 7p22.3p12.3 and duplication 9p24.3p13.2, deletion 3q13.31q22.2 and deletion 3q22.3q23, and deletion 17p13.1 and deletion 1q21.1q21.2. Other two findings were classified as incidental findings (deletion 5q14.3 and 17p12 duplication). Our research shows that 17% of the aberrations (6/35 abnormal results) that cannot be identified by the routine kariotype analysis are structural aberrations containing genes important for fetal development, the mutations of which may cause spontaneous abortion.

Prenatal Diagnosis by Array Comparative Genomic Hybridization in Fetuses with Cardiac Abnormalities.

Congenital heart defects (CHDs) appear in 8-10 out of 1000 live born newborns and are one of the most common causes of deaths. In fetuses, the congenital heart defects are found even 3-5 times more often. Currently, microarray comparative genomic hybridization (array CGH) is recommended by worldwide scientific organizations as a first-line test in the prenatal diagnosis of fetuses with sonographic abnormalities, especially cardiac defects. We present the results of the application of array CGH in 484 cases with prenatally diagnosed congenital heart diseases by fetal ultrasound scanning (256 isolated CHD and 228 CHD coexisting with other malformations). We identified pathogenic aberrations and likely pathogenic genetic loci for CHD in 165 fetuses and 9 copy number variants (CNVs) of unknown clinical significance. Prenatal array-CGH is a useful method allowing the identification of all unbalanced aberrations (number and structure) with a much higher resolution than the currently applied traditional assessment techniques karyotype. Due to this ability, we identified the etiology of heart defects in 37% of cases.

Wide Fontanels, Delayed Speech Development and Hoarse Voice as Useful Signs in the Diagnosis of KBG Syndrome: A Clinical Description of 23 Cases with Pathogenic Variants Involving the ANKRD11 Gene or Submicroscopic Chromosomal Rearrangements of 16q24.3.

KBG syndrome is a neurodevelopmental autosomal dominant disorder characterized by short stature, macrodontia, developmental delay, behavioral problems, speech delay and delayed closing of fontanels. Most patients with KBG syndrome are found to have a mutation in the ANKRD11 gene or a chromosomal rearrangement involving this gene. We hereby present clinical evaluations of 23 patients aged 4 months to 26 years manifesting clinical features of KBG syndrome. Mutation analysis in the patients was performed using panel or exome sequencing and array CGH. Besides possessing dysmorphic features typical of the KBG syndrome, nearly all patients had psychomotor hyperactivity (86%), 81% had delayed speech, 61% had poor weight gain, 56% had delayed closure of fontanel and 56% had a hoarse voice. Macrodontia and a height range of -1 SDs to -2 SDs were noted in about half of the patients; only two patients presented with short stature below -3 SDs. The fact that wide, delayed closing fontanels were observed in more than half of our patients with KBG syndrome confirms the role of the ANKRD11 gene in skull formation and suture fusion. This clinical feature could be key to the diagnosis of KBG syndrome, especially in young children. Hoarse voice is a previously undescribed phenotype of KBG syndrome and could further reinforce clinical diagnosis.

Genetics and epigenetics of autism spectrum disorder-current evidence in the field.

Autism spectrum disorders (ASD) is a heterogenous group of neurodevelopmental disorders characterized by problems in social interaction and communication as well as the presence of repetitive and stereotyped behavior. It is estimated that the prevalence of ASD is 1-2% in the general population with the average male to female ratio 4-5:1. Although the causes of ASD remain largely unknown, the studies have shown that both genetic and environmental factors play an important role in the etiology of these disorders. Array comparative genomic hybridization and whole exome/genome sequencing studies identified common and rare copy number or single nucleotide variants in genes encoding proteins involved in brain development, which play an important role in neuron and synapse formation and function. The genetic etiology is recognized in ~ 25-35% of patients with ASD. In this article, we review the current state of knowledge about the genetic etiology of ASD and also propose a diagnostic algorithm for patients.

A novel de novo 20q13.11q13.12 microdeletion in a boy with neurodevelopmental disorders - case report.

Copy-number variants (CNVs) are an important cause of human neurodevelopmental disorders. We present the first case of a 424 kb de novo 20q13.11q13.12 microdeletion in a patient with attention deficit disorder, tics and autistic behaviors, such as emotional and behavioral problems, and movement stereotypes. This region includes three genes expressed in the brain: SFRS6, PTPRT and L3MBTL. Our results suggest that loss of the chromosomal region 20q13.11q13.12 is causative for the clinical findings observed in the patient.

DYRK1A haploinsufficiency causes a new recognizable syndrome with microcephaly, intellectual disability, speech impairment, and distinct facies.

Dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 A (DYRK1A ) is a highly conserved gene located in the Down syndrome critical region. It has an important role in early development and regulation of neuronal proliferation. Microdeletions of chromosome 21q22.12q22.3 that include DYRK1A (21q22.13) are rare and only a few pathogenic single-nucleotide variants (SNVs) in the DYRK1A gene have been described, so as of yet, the landscape of DYRK1A disruptions and their associated phenotype has not been fully explored. We have identified 14 individuals with de novo heterozygous variants of DYRK1A; five with microdeletions, three with small insertions or deletions (INDELs) and six with deleterious SNVs. The analysis of our cohort and comparison with published cases reveals that phenotypes are consistent among individuals with the 21q22.12q22.3 microdeletion and those with translocation, SNVs, or INDELs within DYRK1A. All individuals shared congenital microcephaly at birth, intellectual disability, developmental delay, severe speech impairment, short stature, and distinct facial features. The severity of the microcephaly varied from -2 SD to -5 SD. Seizures, structural brain abnormalities, eye defects, ataxia/broad-based gait, intrauterine growth restriction, minor skeletal abnormalities, and feeding difficulties were present in two-thirds of all affected individuals. Our study demonstrates that haploinsufficiency of DYRK1A results in a new recognizable syndrome, which should be considered in individuals with Angelman syndrome-like features and distinct facial features. Our report represents the largest cohort of individuals with DYRK1A disruptions to date, and is the first attempt to define consistent genotype-phenotype correlations among subjects with 21q22.13 microdeletions and DYRK1A SNVs or small INDELs.

The usefulness of array comparative genomic hybridization in clinical diagnostics of intellectual disability in children.

INTRODUCTION: Intellectual disability (ID)/Developmental delay (DD), which occurs in 1-3% of the population, accounts for a large number of cases regularly seen in genetics clinics. Currently, Array Comparative Genomic Hybridization (array CGH) is recommended by the International Standards for Cytogenomic Arrays (ISCA) Consortium as a first line test in the diagnostics of ID/DD, replacing G-banded chromosome analysis. THE AIM: Application of array CGH in clinical diagnostics of developmental delay/ intellectual disability in children. MATERIAL AND METHODS: We present the results of 8x60K oligonucleotide array application that was successfully implemented in a cohort of 112 patients with the clinical diagnosis of intellectual disability and accompanying dysmorphic features and/or congenital malformations. RESULTS: We have identified 37 copy number variants (CNVs) with the size ranging from 40 kb to numerical chromosomal aberrations, including unbalanced translocations and chromosome Y disomy, receiving an overall diagnostic yield of 33%. Known pathogenic changes were identified in 21.4% of the cases. Among patients with pathogenic CNVs identified by array CGH, 41.7% had a previously normal karyotype analysis. CONCLUSIONS: Our studies provide more insights into the benefits derived by using chromosomal microarray analysis and demonstrate the usefulness of array CGH as a first-tier clinical setting test in patients with intellectual disability.

Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders.

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.

Application of array comparative genomic hybridization in 256 patients with developmental delay or intellectual disability.

We used whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) in a cohort of 256 patients with developmental delay (DD)/intellectual disability (ID) with or without dysmorphic features, additional neurodevelopmental abnormalities, and/or congenital malformations. In 69 patients, we identified 84 non-polymorphic copy-number variants, among which 41 are known to be clinically relevant, including two recently described deletions, 4q21.21q21.22 and 17q24.2. Chromosomal microarray analysis revealed also 15 potentially pathogenic changes, including three rare deletions, 5q35.3, 10q21.3, and 13q12.11. Additionally, we found 28 copy-number variants of unknown clinical significance. Our results further support the notion that copy-number variants significantly contribute to the genetic etiology of DD/ID and emphasize the efficacy of the detection of novel candidate genes for neurodevelopmental disorders by whole-genome array CGH.

[Genetic bases of autism spectrum disorders]. / Genetyczne Uwarunkowania Zaburzen Autystycznych.

Autism spectrum disorders (ASDs) are an etiologically and clinically heterogeneous group of neurodevelopmental disorders affecting approximately 0.6-1% of the general population. ASDs are characterized by deficits in social communication, impaired language development, and stereotyped repetitive behaviour. The impact of genetic factors in ASDs has been confirmed in the past few years. Numerous studies have shown that among patients with ASDs, approximately 10% have DNA copy number variation and 10-20% point mutations. Most of the deficiencies identified in individuals with ASDs relate to genes encoding proteins involved mainly in the development of neurons and their synapses functioning in various signaling pathways. Due to the large heterogeneity of identified changes in the genome of individuals with ASDs, the newest techniques enabling analysis of the entire genome in one study (microarrays, next-generation sequencing) are the methods of choice in the diagnostics of this pathology.

Multiple samples aCGH analysis for rare CNVs detection.

BACKGROUND: DNA copy number variations (CNV) constitute an important source of genetic variability. The standard method used for CNV detection is array comparative genomic hybridization (aCGH). RESULTS: We propose a novel multiple sample aCGH analysis methodology aiming in rare CNVs detection. In contrast to the majority of previous approaches, which deal with cancer datasets, we focus on constitutional genomic abnormalities identified in a diverse spectrum of diseases in human. Our method is tested on exon targeted aCGH array of 366 patients affected with developmental delay/intellectual disability, epilepsy, or autism. The proposed algorithms can be applied as a post-processing filtering to any given segmentation method. CONCLUSIONS: Thanks to the additional information obtained from multiple samples, we could efficiently detect significant segments corresponding to rare CNVs responsible for pathogenic changes. The robust statistical framework applied in our method enables to eliminate the influence of widespread technical artifact termed 'waves'.

Application of custom-designed oligonucleotide array CGH in 145 patients with autistic spectrum disorders.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders, including childhood autism, atypical autism, and Asperger syndrome, with an estimated prevalence of 1.0-2.5% in the general population. ASDs have a complex multifactorial etiology, with genetic causes being recognized in only 10-20% of cases. Recently, copy-number variants (CNVs) have been shown to contribute to over 10% of ASD cases. We have applied a custom-designed oligonucleotide array comparative genomic hybridization with an exonic coverage of over 1700 genes, including 221 genes known to cause autism and autism candidate genes, in a cohort of 145 patients with ASDs. The patients were classified according to ICD-10 standards and the Childhood Autism Rating Scale protocol into three groups consisting of 45 individuals with and 69 individuals without developmental delay/intellectual disability (DD/ID), and 31 patients, in whom DD/ID could not be excluded. In 12 patients, we have identified 16 copy-number changes, eight (5.5%) of which likely contribute to ASDs. In addition to known recurrent CNVs such as deletions 15q11.2 (BP1-BP2) and 3q13.31 (including DRD3 and ZBTB20), and duplications 15q13.3 and 16p13.11, our analysis revealed two novel genes clinically relevant for ASDs: ARHGAP24 (4q21.23q21.3) and SLC16A7 (12q14.1). Our results further confirm the diagnostic importance of array CGH in detection of CNVs in patients with ASDs and demonstrate that CNVs are an important cause of ASDs as a heterogeneous condition with a variety of contributory genes.

Application of array comparative genomic hybridization in 102 patients with epilepsy and additional neurodevelopmental disorders.

Copy-number variants (CNVs) collectively represent an important cause of neurodevelopmental disorders such as developmental delay (DD)/intellectual disability (ID), autism, and epilepsy. In contrast to DD/ID, for which the application of microarray techniques enables detection of pathogenic CNVs in -10-20% of patients, there are only few studies of the role of CNVs in epilepsy and genetic etiology in the vast majority of cases remains unknown. We have applied whole-genome exon-targeted oligonucleotide array comparative genomic hybridization (array CGH) to a cohort of 102 patients with various types of epilepsy with or without additional neurodevelopmental abnormalities. Chromosomal microarray analysis revealed 24 non-polymorphic CNVs in 23 patients, among which 10 CNVs are known to be clinically relevant. Two rare deletions in 2q24.1q24.3, including KCNJ3 and 9q21.13 are novel pathogenic genetic loci and 12 CNVs are of unknown clinical significance. Our results further support the notion that rare CNVs can cause different types of epilepsy, emphasize the efficiency of detecting novel candidate genes by whole-genome array CGH, and suggest that the clinical application of array CGH should be extended to patients with unexplained epilepsies.

Assessment of the role of copy-number variants in 150 patients with congenital heart defects.

BACKGROUND: Congenital heart defects are the most common group of major birth anomalies and one of the leading causes of infant deaths. Mendelian and chromosomal syndromes account for about 20% of congenital heart defects and in some cases are associated with other malformations, intellectual disability, and/or dysmorphic features. The remarkable conservation of genetic pathways regulating heart development in animals suggests that genetic factors can be responsible for a significantly higher percentage of cases. THE AIM: Assessment of the role of CNVs in the etiology of congenital heart defects using microarray studies. MATERIAL AND METHODS: Genome-wide array comparative genomic hybridization, targeting genes known to play an important role in heart development or responsible for abnormal cardiac phenotype was used in the study on 150 patients. In addition, we have used multiplex ligation-dependent probe amplification specific for chromosome 22q11.2 region. RESULTS: We have identified 21 copy-number variants, including 13 known causative recurrent rearrangements (12 deletions 22q11.2 and one deletion 7q11.23), three potentially pathogenic duplications (5q14.2, 15q13.3, and 22q11.2), and five variants likely benign for cardiac anomalies. We suggest that abnormal copy-number of the ARRDC3 and KLF13 genes can be responsible for heart defects. CONCLUSIONS: Our study demonstrates that array comparative genomic hybridization enables detection of clinically significant chromosomal imbalances in patients with congenital heart defects.

Detection of clinically relevant exonic copy-number changes by array CGH.

Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

Intragenic rearrangements in NRXN1 in three families with autism spectrum disorder, developmental delay, and speech delay.

NRXN1 is highly expressed in brain and has been shown recently to be associated with ASD, schizophrenia, cognitive and behavioral abnormalities, and alcohol and nicotine dependence. We present three families, in whom we identified intragenic rearrangements within NRXN1 using a clinical targeted oligonucleotide array CGH. An approximately 380 kb deletion was identified in a woman with Asperger syndrome, anxiety, and depression and in all four of her children affected with autism, anxiety, developmental delay, and speech delay but not in an unaffected child. An approximately 180 kb tandem duplication was found in a patient with autistic disorder and cognitive delays, and in his mother and younger brother who have speech delay. An approximately 330 kb tandem duplication was identified in a patient with autistic features. As predicted by conceptual translation, all three genomic rearrangements led to the premature truncation of NRXN1. Our data support previous observations that NRXN1 may be pathogenic in a wide variety of psychiatric diseases, including autism spectrum disorder, global developmental delay, anxiety, and depression.

Rare pathogenic microdeletions and tandem duplications are microhomology-mediated and stimulated by local genomic architecture.

Genomic copy number variation (CNV) plays a major role in various human diseases as well as in normal phenotypic variability. For some recurrent disease-causing CNVs that convey genomic disorders, the causative mechanism is meiotic, non-allelic, homologous recombination between breakpoint regions exhibiting extensive sequence homology (e.g. low-copy repeats). For the majority of recently identified rare pathogenic CNVs, however, the mechanism is unknown. Recently, a model for CNV formation implicated mitotic replication-based mechanisms, such as (alternative) non-homologous end joining and fork stalling and template switching, in the etiology of human pathogenic CNVs. The extent to which such mitotic mechanisms contribute to rare pathogenic CNVs remains to be determined. In addition, it is unexplored whether genomic architectural features such as repetitive elements or sequence motifs associated with DNA breakage stimulate the formation of rare pathogenic CNVs. To this end, we have sequenced breakpoint junctions of 30 rare pathogenic microdeletions and eight tandem duplications, representing the largest series of such CNVs examined to date in this much detail. Our results demonstrate the presence of (micro)homology ranging from 2 to over 75 bp, in 79% of the breakpoint junctions. This indicates that microhomology-mediated repair mechanisms, including the recently reported fork stalling and template switching and/or microhomology-mediated break-induced replication, prevail in rare pathogenic CNVs. In addition, we found that the vast majority of all breakpoints (81%) were associated with at least one of the genomic architectural features evaluated. Moreover, 75% of tandem duplication breakpoints were associated with the presence of one of two novel sequence motifs. These data suggest that rare pathogenic microdeletions and tandem duplications do not occur at random genome sequences, but are stimulated and potentially catalyzed by various genomic architectural features.

Clinical and molecular-cytogenetic evaluation of a family with partial Jacobsen syndrome without thrombocytopenia caused by an approximately 5 Mb deletion del(11)(q24.3).

Clinical manifestations of Jacobsen syndrome (JBS) depend on the size of the 11qter deletion, which usually varies between approximately 7 and 20 Mb. Typical JBS features include developmental delay/mental retardation, short stature, congenital heart defects, thrombocytopenia, and characteristic dysmorphic facial features. We report on a family in which a 4-year-old girl as well as her mother and maternal uncle present with subtle features of JBS. Notably, neither thrombocytopenia nor congenital anomalies were detected in this family. Cytogenetic analyses revealed normal karyotypes. Using fluorescence in situ hybridization (FISH) and whole-genome oligonucleotide array CGH analyses, we identified an approximately 5 Mb deletion of the terminal part of chromosome 11q in all the three affected family members. The deletion breakpoint was mapped between 129,511,419 and 129,519,794 bp. This is the smallest deletion reported in a JBS patient. Interestingly, the FLI1 (friend leukemia virus integration 1) hematopoiesis factor gene located approximately 6.5 Mb from 11qter and usually deleted in patients with JBS, is intact. Our data support previous hypotheses that FLI1 haploinsufficiency is responsible for thrombocytopenia in patients with JBS.