Nancy B. Spinner

Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Mutations in the human Jagged1 gene are responsible for Alagille syndrome

Alagille syndrome (AGS) is an autosomal-dominant disorder characterized by intrahepatic cholestasis and abnormalities of heart, eye and vertebrae, as well as a characteristic facial appearance. Identification of rare AGS patients with cytogenetic deletions has allowed mapping of the gene to 20p12. We have generated a cloned contig of the critical region and used fluorescent in situ hybridization on cells from patients with submicroscopic deletions to narrow the candidate region to only 250 kb. Within this region we identified JAG1, the human homologue of rat Jagged1, which encodes a ligand for the Notch receptor. Cell-cell Jagged/Notch interactions are known to be critical for determination of cell fates in early development, making this an attractive candidate gene for a developmental disorder in humans. Determining the complete exon–intron structure of JAG1 allowed detailed mutational analysis of DMA samples from non-deletion AGS patients, revealing three frame-shift mutations, two splice donor mutations and one mutation abolishing RNA expression from the altered allele. We conclude that AGS is caused by haploinsufficiency of JAG1.

Microduplications of 16p11.2 are associated with schizophrenia.

Recurrent microdeletions and microduplications of a 600-kb genomic region of chromosome 16p11.2 have been implicated in childhood-onset developmental disorders. We report the association of 16p11.2 microduplications with schizophrenia in two large cohorts. The microduplication was detected in 12/1,906 (0.63%) cases and 1/3,971 (0.03%) controls (P = 1.2 × 10−5, OR = 25.8) from the initial cohort, and in 9/2,645 (0.34%) cases and 1/2,420 (0.04%) controls (P = 0.022, OR = 8.3) of the replication cohort. The 16p11.2 microduplication was associated with a 14.5-fold increased risk of schizophrenia (95% CI (3.3, 62)) in the combined sample. A meta-analysis of datasets for multiple psychiatric disorders showed a significant association of the microduplication with schizophrenia (P = 4.8 × 10−7), bipolar disorder (P = 0.017) and autism (P = 1.9 × 10−7). In contrast, the reciprocal microdeletion was associated only with autism and developmental disorders (P = 2.3 × 10−13). Head circumference was larger in patients with the microdeletion than in patients with the microduplication (P = 0.0007).

Points to Consider: Ethical, Legal, and Psychosocial Implications of Genetic Testing in Children and Adolescents

In 1995, the American Society of Human Genetics (ASHG) and American College of Medical Genetics and Genomics (ACMG) jointly published a statement on genetic testing in children and adolescents. In the past 20 years, much has changed in the field of genetics, including the development of powerful new technologies, new data from genetic research on children and adolescents, and substantial clinical experience. This statement represents current opinion by the ASHG on the ethical, legal, and social issues concerning genetic testing in children. These recommendations are relevant to families, clinicians, and investigators. After a brief review of the 1995 statement and major changes in genetic technologies in recent years, this statement offers points to consider on a broad range of test technologies and their applications in clinical medicine and research. Recommendations are also made for record and communication issues in this domain and for professional education.

NOTCH2 Mutations Cause Alagille Syndrome, a Heterogeneous Disorder of the Notch Signaling Pathway

Alagille syndrome (AGS) is caused by mutations in the gene for the Notch signaling pathway ligand Jagged1 (JAG1), which are found in 94% of patients. To identify the cause of disease in patients without JAG1 mutations, we screened 11 JAG1 mutation-negative probands with AGS for alterations in the gene for the Notch2 receptor (NOTCH2). We found NOTCH2 mutations segregating in two families and identified five affected individuals. Renal manifestations, a minor feature in AGS, were present in all the affected individuals. This demonstrates that AGS is a heterogeneous disorder and implicates NOTCH2 mutations in human disease.

High-resolution mapping and analysis of copy number variations in the human genome: A data resource for clinical and research applications

We present a database of copy number variations (CNVs) detected in 2026 disease-free individuals, using high-density, SNP-based oligonucleotide microarrays. This large cohort, comprised mainly of Caucasians (65.2%) and African-Americans (34.2%), was analyzed for CNVs in a single study using a uniform array platform and computational process. We have catalogued and characterized 54,462 individual CNVs, 77.8% of which were identified in multiple unrelated individuals. These nonunique CNVs mapped to 3272 distinct regions of genomic variation spanning 5.9% of the genome; 51.5% of these were previously unreported, and >85% are rare. Our annotation and analysis confirmed and extended previously reported correlations between CNVs and several genomic features such as repetitive DNA elements, segmental duplications, and genes. We demonstrate the utility of this data set in distinguishing CNVs with pathologic significance from normal variants. Together, this analysis and annotation provides a useful resource to assist with the assessment of CNVs in the contexts of human variation, disease susceptibility, and clinical molecular diagnostics.

Mechanisms of mosaicism, chimerism and uniparental disomy identified by single nucleotide polymorphism array analysis

Mosaic aneuploidy and uniparental disomy (UPD) arise from mitotic or meiotic events. There are differences between these mechanisms in terms of (i) impact on embryonic development; (ii) co-occurrence of mosaic trisomy and UPD and (iii) potential recurrence risks. We used a genome-wide single nucleotide polymorphism (SNP) array to study patients with chromosome aneuploidy mosaicism, UPD and one individual with XX/XY chimerism to gain insight into the developmental mechanism and timing of these events. Sixteen cases of mosaic aneuploidy originated mitotically, and these included four rare trisomies and all of the monosomies, consistent with the influence of selective factors. Five trisomies arose meiotically, and three of the five had UPD in the disomic cells, confirming increased risk for UPD in the case of meiotic non-disjunction. Evidence for the meiotic origin of aneuploidy and UPD was seen in the patterns of recombination visible during analysis with 1-3 crossovers per chromosome. The mechanisms of formation of the UPD included trisomy rescue, with and without concomitant trisomy, monosomy rescue, and mitotic formation of a mosaic segmental UPD. UPD was also identified in an XX/XY chimeric individual, with one cell line having complete maternal UPD consistent with a parthenogenetic origin. Utilization of SNP arrays allows simultaneous evaluation of genomic alterations and insights into aneuploidy and UPD mechanisms. Differentiation of mitotic and meiotic origins for aneuploidy and UPD supports existence of selective factors against full trisomy of some chromosomes in the early embryo and provides data for estimation of recurrence and disease mechanisms.

Vascular anomalies in Alagille syndrome: a significant cause of morbidity and mortality.

Background—Alagille syndrome (AGS) is a dominantly inherited multisystem disorder involving the liver, heart, eyes, face, and skeleton, caused by mutations in Jagged1. Intracranial bleeding is a recognized complication and cause of mortality in AGS. There are multiple case reports of intracranial vessel abnormalities and other vascular anomalies in AGS. The objective of this study was to characterize the nature and spectrum of vascular anomalies in AGS. Methods and Results—Retrospective chart review of 268 individuals with AGS was performed. Twenty-five patients (9%) had noncardiac vascular anomalies or events. Sixteen patients had documented structural vascular abnormalities. Two had basilar artery aneurysms, 7 had internal carotid artery anomalies, and another had a middle cerebral artery aneurysm. Moyamoya disease was described in 1 patient. Three of the 16 patients had aortic aneurysms, and 2 had aortic coarctations. One of the patients with a basilar artery aneurysm also had coarctation of the aorta. One of the individuals with an internal carotid artery anomaly also had renal artery stenosis. Nine more patients had intracranial events without documented vessel abnormalities. Vascular accidents accounted for 34% of the mortality in this cohort. Conclusions—The vascular anomalies described in our cohort of AGS individuals identify an underrecognized and potentially devastating complication of this disorder. It is a major cause of morbidity and mortality in this population, accounting for 34% of the mortality. We have also reviewed the body of evidence supporting a role for Jagged1 and the Notch signaling pathway in vascular development.

A genomic view of mosaicism and human disease

Genomic technologies, including next-generation sequencing (NGS) and single-nucleotide polymorphism (SNP) microarrays, have provided unprecedented opportunities to assess genomic variation among, and increasingly within, individuals. It has long been known that cancer is a mosaic genetic disorder, but mosaicism is now apparent in a diverse range of other clinical disorders, as indicated by their tissue distributions and inheritance patterns. Recent technical advances have uncovered the causative mosaic variant underlying many of these conditions and have provided insight into the pervasiveness of mosaicism in normal individuals. Here, we discuss the clinical and molecular classes of mosaicism, their detection and the biological insights gained from these studies.

Analysis of Cardiovascular Phenotype and Genotype-Phenotype Correlation in Individuals With a JAG1 Mutation and/or Alagille Syndrome

Background—Cardiovascular anomalies are among the most common features of Alagille syndrome (AGS). Mutations of JAG1 are found in most individuals with AGS. This study was undertaken to determine the spectrum of cardiovascular phenotypes associated with a JAG1 mutation and/or AGS, investigate potential genotype-phenotype correlations, and begin to correlate clinical outcome with genetic pathogenesis. Methods and Results—We reviewed the records of 200 individuals with a JAG1 mutation or AGS. A total of 187 (94%) subjects had evidence of cardiovascular involvement. Cardiovascular anomalies were identified by imaging in 150 subjects (75%), and 37 (19%) had a peripheral pulmonary stenosis murmur with either a normal echocardiogram or no imaging study. Of the 150 subjects with anomalies confirmed by imaging, right-sided anomalies were present in 123 and left-sided anomalies in 22, with both in 12. Seventeen subjects had other anomalies. The most common abnormality was stenosis/hypoplasia of the branch pulmonary arteries (PAs), which was documented by imaging (n=111) or inferred from a peripheral pulmonary stenosis murmur (n=41) in 76% of subjects. Tetralogy of Fallot was present in 23 subjects and was accompanied by pulmonary atresia in 8. Branch PA phenotype differed between individuals with and without a JAG1 mutation. Among subjects with a JAG1 mutation, there was no correlation between the type or location of mutation and the frequency or type of cardiovascular anomaly. Conclusions—More than 90% of individuals with a JAG1 mutation or AGS have cardiovascular anomalies, with branch PA stenosis the most common abnormality. Cardiovascular phenotype does not correlate with the type or location of JAG1 mutation.