N. de Leeuw

Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome

Background: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. Methods: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3–BP4–BP5 region were included in this study to ascertain the clinical significance of duplications in this region. Results: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3–BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3–BP4–BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. Conclusions: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

Identification of clinically significant, submicroscopic chromosome alterations and UPD in fetuses with ultrasound anomalies using genome-wide 250k SNP array analysis

Background The implementation of microarray analysis in prenatal diagnostics is a topic of discussion, as rare copy number variants with unknown/uncertain clinical consequences are likely to be found. The application of targeted microarrays limits such findings, but the potential disadvantage is that relevant, so far unknown, aberrations might be overlooked. Therefore, we explore the possibilities for the prenatal application of the genome-wide 250k single nucleotide polymorphism array platform. Methods Affymetrix 250k NspI single nucleotide polymorphism array analysis (Affymetrix, Inc., Santa Clara, California, USA) was performed on DNA from 38 prenatally karyotyped fetuses with ultrasound anomalies. Analyses were performed after termination of pregnancy, intrauterine fetal death or birth on DNA isolated from fetal or neonatal material. Results Aberrations were detected in 17 of 38 fetuses, 6 of whom with a previously identified chromosomal abnormality and 11 with previously normal or balanced karyotypes. Of the latter, the detected aberration occurred de novo and was considered of clinical relevance in five cases (16%), inherited from a healthy parent in four cases (12%), and de novo yet with unclear clinical relevance in two cases (6%). The clinically relevant abnormalities either were novel copy number variants (n=3) or concerned a uniparental disomy (n=2). Conclusion In at least 16% of fetuses with ultrasound anomalies and a normal or balanced karyotype, causal (submicroscopic) aberrations were detected, illustrating the importance of the (careful) implementation of microarray analysis in prenatal diagnosis. The fact that the identified, clinically relevant, aberrations would have gone undetected with most targeted approaches underscores the added value of a genome-wide approach.

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.

De novo copy number variants associated with intellectual disability have a paternal origin and age bias

Background De novo mutations and structural rearrangements are a common cause of intellectual disability (ID) and other disorders with reduced or null reproductive fitness. Insight into the genomic and environmental factors predisposing to the generation of these de novo events is therefore of significant clinical importance. Methods This study used information from single nucleotide polymorphism microarrays to determine the parent-of-origin of 118 rare de novo copy number variations (CNVs) detected in a cohort of 3443 patients with ID. Results The large majority of these CNVs (76%, p=1.14×10−8) originated on the paternal allele. This paternal bias was independent of CNV length and CNV type. Interestingly, the paternal bias was less pronounced for CNVs flanked by segmental duplications (64%), suggesting that molecular mechanisms involved in the formation of rare de novo CNVs may be dependent on the parent-of-origin. In addition, a significantly increased paternal age was only observed for those CNVs which were not flanked by segmental duplications (p=0.02). Conclusion This indicates that rare de novo CNVs are increasingly being generated with advanced paternal age by replication based mechanisms during spermatogenesis.

Clinical significance of de novo and inherited copy-number variation.

Copy‐number variations (CNVs) are a common cause of intellectual disability and/or multiple congenital anomalies (ID/MCA). However, the clinical interpretation of CNVs remains challenging, especially for inherited CNVs. Well‐phenotyped patients (5,531) with ID/MCA were screened for rare CNVs using a 250K single‐nucleotide polymorphism array platform in order to improve the understanding of the contribution of CNVs to a patients phenotype. We detected 1,663 rare CNVs in 1,388 patients (25.1%; range 0–5 per patient) of which 437 occurred de novo and 638 were inherited. The detected CNVs were analyzed for various characteristics, gene content, and genotype–phenotype correlations. Patients with severe phenotypes, including organ malformations, had more de novo CNVs (P < 0.001), whereas patient groups with milder phenotypes, such as facial dysmorphisms, were enriched for both de novo and inherited CNVs (P < 0.001), indicating that not only de novo but also inherited CNVs can be associated with a clinically relevant phenotype. Moreover, patients with multiple CNVs presented with a more severe phenotype than patients with a single CNV (P < 0.001), pointing to a combinatorial effect of the additional CNVs. In addition, we identified 20 de novo single‐gene CNVs that directly indicate novel genes for ID/MCA, including ZFHX4, ANKH, DLG2, MPP7, CEP89, TRIO, ASTN2, and PIK3C3.

Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: A multicenter study†

Genomic microarrays have been implemented in the diagnosis of patients with unexplained mental retardation. This method, although revolutionizing cytogenetics, is still limited to the detection of rare de novo copy number variants (CNVs). Genome‐wide single nucleotide polymorphism (SNP) microarrays provide high‐resolution genotype as well as CNV information in a single experiment. We hypothesize that the widespread use of these microarray platforms can be exploited to greatly improve our understanding of the genetic causes of mental retardation and many other common disorders, while already providing a robust platform for routine diagnostics. Here we report a detailed validation of Affymetrix 500k SNP microarrays for the detection of CNVs associated to mental retardation. After this validation we applied the same platform in a multicenter study to test a total of 120 patients with unexplained mental retardation and their parents. Rare de novo CNVs were identified in 15% of cases, showing the importance of this approach in daily clinical practice. In addition, much more genomic variation was observed in these patients as well as their parents. We provide all of these data for the scientific community to jointly enhance our understanding of these genomic variants and their potential role in this common disorder. Hum Mutat 30:1–11, 2009.

Intragenic deletion in DYRK1A leads to mental retardation and primary microcephaly

To the Editor : For years, human genotype–phenotype studies have suggested DYRK1A (MIM 600855) as a candidate gene for microcephaly and mental retardation (MR) (1–3). These findings have been supported by various animal models such as Drosophila and mice (4, 5). DYRK1A is a member of the dual-specificity tyrosine phosphorylationregulated kinase (DYRK) family and participates in various cellular processes. It is a highly conserved gene located in the Down Syndrome Critical Region, a part on chromosome 21 that is responsible for the majority of phenotypic features in Down syndrome (6, 7). Convincing evidence for a brain developmental function of DYRK1A in humans was recently provided by a report on two mentally retarded individuals with a balanced translocation truncating this gene (8). However, so far, no pure DYRK1A deletions in humans have been reported. In this study, we investigated whether smaller copy number variations (CNVs), below the routine diagnostic threshold set at 150 kb, may be present at the DYRK1A locus. Therefore, the intensity log2 ratios of all single nucleotide polymorphism (SNP) probes located within and surrounding the DYRK1A gene (37,647,772–37,813,457 bp) were studied in 3009 mentally retarded individuals, in whom no pathogenic CNVs (>150 kb) were detected during routine diagnostic 250K SNP array analysis (Affymetrix Inc., Santa Clara, CA) (9). Patients in which at least four consecutive SNP probes showed an abnormal log2 ratio (<−0.2 or >0.25) were considered to be candidates for having a DYRK1A deletion or duplication. Of the 16 candidate aberrations fulfilling this criterion one deletion could be confirmed by quantitative polymerase chain reaction (qPCR, Supporting information, Table S1) and was shown to be de novo (Fig. 1b). A high-resolution NimbleGen hg18 chromosome 21 specific 385K array subsequently revealed that the deletion had a size of 52 kb (37,796,500–37,849,000 bp) and deleted the last three exons of DYRK1A (Fig. 1c). The female with the DYRK1A deletion was born with a length of 48 cm (−2 SD), weight of 3195 g (−1 SD) and a head circumference of 33 cm (<−2 SD). From birth onward, she had feeding problems with failure to thrive and non-fluent motoric movements and hypoactivity were noted. During infancy she regularly had febrile seizures. At 24 years of age, a cerebral magnetic resonance imaging (MRI) showed a mild atrophic brain and medulla without structural congenital anomalies. At 33 years of age, she had severe MR without speech and showed anxious autistic behavior. She walked with a stiff motoric gait and jerky movements. She had a slender posture with a length of 161.5 cm (−1.5 SD) and an occipitofrontal circumference (OFC) of 50.5 cm (−3 SD). Facial dysmorphisms included bitemporal narrowing, deep-set eyes, large simple ears and a pointed nasal tip. In addition, she had eczema, hypoplasia of breasts, hallux valgus of feet and an irregular implant of toes. Several features in this patient, such as severe developmental delay, growth retardation, nonfluent motoric movements, microcephaly in the absence of structural brain anomalies, hypoactivity and anxious behavior, show a striking similarity with features observed in different animal models with mutated DYRK1A. The Drosophila ortholog of DYRK1A is the minibrain (mnb) gene (5). The mnb gene plays an important role in regulating the number of distinct neuronal cell types. These mutants are slightly smaller in size and show behavior problems, characterized by poor visual pattern fixation, reduced walking activity and speed, and poor odor discrimination (5). Dyrk1A+/− mice show preand postnatal growth retardation, developmental delay, specific motor and learning deficits, visual impairment, behavioral defects and increased anxiety (4, 10–13). Brains of mutant mnb flies and Dyrk1A+/− mice are greatly reduced in size, although the neuronal architecture remains unchanged. On the basis of human genotype–phenotype studies of partial monosomies 21, DYRK1A has

Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes

Infantile juvenile polyposis is a rare disease with severe gastrointestinal symptoms and a grave clinical course. Recently, 10q23 microdeletions involving the PTEN and BMPR1A genes were found in four patients with infantile juvenile polyposis. It was hypothesized that a combined and synergistic effect of the deletion of both genes would explain the condition. Subsequently, however, a patient with a larger 10q23 deletion including the same genes but with a mild clinical phenotype was identified. Here, we present four additional patients with 10q23 microdeletions involving the PTEN and BMPR1A genes. The sizes of the deletions were analyzed using single nucleotide polymorphism array analysis. All patients had macrocephaly, dysmorphic features, retardation and congenital abnormalities. One patient developed colorectal cancer. However, only one case had disease onset before 2 years of age and severe symptoms requiring colectomy. No clear correlation was found between ages at onset or severity of gastrointestinal symptoms and the sizes of the deletions. We conclude that patients with 10q23 microdeletions involving the PTEN and BMPR1A genes have variable clinical phenotypes, which cannot be explained merely by the deletion sizes. The phenotypes are not restricted to severe infantile juvenile polyposis but include childhood‐onset cases with macrocephaly, retardation, mild gastrointestinal symptoms and possibly early‐onset colorectal cancer.

Pathogenic or not? Assessing the clinical relevance of copy number variants

The availability of commercially produced genomic microarrays has resulted in the wide spread implementation of genomic microarrays, often as a first‐tier diagnostic test for copy number variant (CNV) screening of patients who are suspected for chromosomal aberrations. Patients with intellectual disability (ID) and/or multiple congenital anomalies (MCA) were traditionally the main focus for this microarray‐based CNV screening, but the application of microarrays to other (neurodevelopmental) disorders and tumor diagnostics has also been explored and implemented. The diagnostic workflow for patients with ID is now well established, relying on the identification of rare CNVs and determining their inheritance patterns. However, experience gained through screening large numbers of samples has revealed many subtleties and complexities of CNV interpretation. This has resulted in a better understanding of the contribution of CNVs to genomic disorders not only via de novo occurrence, but also via X‐linked and recessive inheritance models as well as through models taking into account mosaicisms, imprinting, and digenic inheritance. In this review, we discuss CNV interpretation within the context of these different genetic disease models and common pitfalls that can occur when searching for supportive evidence that a CNV is clinically relevant.

A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features

Background Congenital deletions affecting 3q11q23 have rarely been reported and only five cases have been molecularly characterised. Genotype—phenotype correlation has been hampered by the variable sizes and breakpoints of the deletions. In this study, 14 novel patients with deletions in 3q11q23 were investigated and compared with 13 previously reported patients. Methods Clinical data were collected from 14 novel patients that had been investigated by high resolution microarray techniques. Molecular investigation and updated clinical information of one cytogenetically previously reported patient were also included. Results The molecular investigation identified deletions in the region 3q12.3q21.3 with different boundaries and variable sizes. The smallest studied deletion was 580 kb, located in 3q13.31. Genotype—phenotype comparison in 24 patients sharing this shortest region of overlapping deletion revealed several common major characteristics including significant developmental delay, muscular hypotonia, a high arched palate, and recognisable facial features including a short philtrum and protruding lips. Abnormal genitalia were found in the majority of males, several having micropenis. Finally, a postnatal growth pattern above the mean was apparent. The 580 kb deleted region includes five RefSeq genes and two of them are strong candidate genes for the developmental delay: DRD3 and ZBTB20. Conclusion A newly recognised 3q13.31 microdeletion syndrome is delineated which is of diagnostic and prognostic value. Furthermore, two genes are suggested to be responsible for the main phenotype.