Ian D. Krantz

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.

Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for notch1

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational f rameshifts, resulting in gross alterations of the protein product. Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

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.

Rare variants create synthetic genome-wide associations.

A large number of different common variants has been associated with very modest increases of risk for various common diseases. A simulation study shows that rare variants with much greater impacts on disease risk may be responsible for some of these associations.

Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

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).

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.

Mutations in Cohesin Complex Members SMC3 and SMC1A Cause a Mild Variant of Cornelia de Lange Syndrome with Predominant Mental Retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (∼5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

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.