Case 1: Ms. Sanfilippo is a 25-year-old gravida 5 para 3104 whose first-trimester screen gave her a 1:1200 risk of Down syndrome and <1:5000 risk of trisomy 18, and a second-trimester alpha fetoprotein was in the normal range (1.2 MOM). She has no significant past medical history. Her obstetric history was significant for a previous term spontaneous vaginal delivery complicated by a rectovaginal fistula. This was followed by a term cesarean delivery, and her last pregnancy was a preterm repeat cesarean at 34 weeks. A three generation family history was significant for Hirschsprung disease in a son with a previous partner. An initial anatomy ultrasound at 18 weeks of gestation was performed at an outside hospital, and showed the fetus to have a left sided diaphragmatic hernia with cardiac displacement to the right side of the chest. The patient was referred to a regional center because of the abnormal ultrasound. A repeat ultrasound at 20 weeks of gestation identified a thickened nuchal fold of 7 mm, bilateral echogenic kidneys with nephromegaly, and a left-sided diaphragmatic hernia. Ms. Sanfilippo was counseled about the risks and benefits of amniocentesis. Rapid FISH analysis was performed on 50 interphase cells from the amniotic fluid with #13, #18, #21, X-, and Y-specific probes. No evidence of trisomy 13, 18, or 21 was detected. Analysis of the X and Y probe revealed two copies of the X probe and no copies of the Y probe, consistent with 46,XX female karyotype. FISH analysis was also performed using probes specific for chromosome 12 to rule out mosaic isochromosome 12p (Pallister-Killian syndrome), and the results were consistent with normal chromosome 12. Fifteen cells were analyzed from the amniotic fluid specimen. No significant numerical or structural aberrations were seen at the 475 G-band level of resolution. The karyotype was reported as normal, 46,XX female. Since the karyotype and FISH results were both normal, an oligonucleotide microarray was performed. Microarray-based comparative genomic hybridization (aCGH), also known as chromosomal microarray technique (CMA) was performed using a 135K-feature whole-genome microarray. Microarray revealed a 1.4-Mb deletion on 17q12. Neither parent carried the deletion; therefore this deletion was a de novo finding. The deletion caused haploinsufficiency for 17 genes, including AATF, ACACA, DDX52, DUSP14, GGNBP2, HNF1B, LHX1, PIGW, SYNRG, TADA2A, and ZNHIT3. The deleted region on 17q12 is similar in size and gene content to the previously reported 17q12 microdeletion syndrome.1 The 17q12 microdeletion syndrome has been associated with MODY5 (maturity-onset of diabetes of the young, type 5), cystic renal disease, pancreatic atrophy, liver abnormalities, cognitive impairment, and structural brain abnormalities. Ms. Sanfilippo was counseled with regards to the diagnosis and prognosis. The risk of recurrence is at most 2% to 3%, as gonadal mosaicism cannot be ruled out. After reviewing the risks and prognosis associated with congenital diaphragmatic hernia and 17q12 microdeletion syndrome, Ms. Sanfilippo opted for comfort measures after her baby is born.
Chromosomal abnormalities occur in germ cell division (meiosis), during early fetal development, or after birth in any cell in the body (mitosis) (see Chapter 2). The number, structure, properties of chromosomes, chromosomal behavior, and influence of chromosomal abnormalities on the phenotype are studied in cytogenetics. The analysis of chromosomes in human development and disease is accomplished through classical cytogenetic procedures, including Giemsa or G-banding karyotype analysis and C-(constitutive heterochromatin) banding. Molecular techniques such as array comparative genomic hybridization (aCGH/CMA) and single nucleotide polymorphism microarray (SNP microarray) analyses have improved resolution, and have replaced the karyotype in many clinical situations as the first line technique to detect genomic imbalances.
G-BANDED KARYOTYPE ANALYSIS
Cells duplicate their genetic material and generate genetically identical daughter cells during mitosis. Chromosome morphology can be visualized and studied under the light microscope during the prophase or metaphase of the mitotic cell division, when chromosomes are condensed. During metaphase, replicated chromosomes are aligned and sister chromatids are ready for separation to the opposite poles along the spindle fibers. Colchicine, a spindle inhibitor, added to the cell culture, disrupts the spindle-fiber complex and arrests mitosis. The chromosomes, when stained with a dye (Giemsa), have a distinct banded pattern (G-banding) that provides a unique bar code to each chromosome (Figure 15-1). Chromosomes become gradually shortened as the cell cycle progresses from interphase to metaphase. Depending on the degree of chromosome condensation, the chromosomes reveal from 400 to 850 bands per haploid genome, which enables detection of chromosomal alterations with resolution to the 5 to 10 Mb level by routine microscopic analysis. High-resolution chromosome studies (600-850 bands) allow a more detailed analysis of the chromosome structure, as compared to the 400 to 550 bands observed with routine metaphase banding. The karyotype provides an overview of the whole genome and detects both numerical and gross structural chromosomal aberrations.
A human normal male karyotype. Homologous chromosomes (homologues), the two chromosomes in a pair of autosomes, are composed of similar (but not identical) DNA sequences. Each homologue encodes the same set of genes in the same order, but may contain different variant forms of the same gene (allele), as well as variable noncoding DNA (introns). Centromeres are indicated by the dashed lines, separating the short and long arms.
Karyotyping requires viable tissue samples to establish cell culture, and induce mitosis, followed by cell harvesting, chromosome banding, and microscopic analysis. White blood cells, particularly T-lymphocytes, stimulated by phytohemagglutinin (PHA), rapidly divide during 48 to 72 hours of incubation and produce good quality high-resolution metaphases. In short-term blood cultures, mitogen-stimulated cells divide from one to five times, while tissues derived from chorionic villi, amniocytes, and fibroblasts undergo 20 to 100 mitotic cell divisions. Because of numerous cell divisions, some cells may undergo abnormal division and introduce a cell line with abnormal chromosome structure or number. These in vitro cultural artifacts lead to pseudomosaicism (see Chapter 2). Minimizing the time of a sample in culture provides cytogenetic results that most closely reflect in vivo conditions.
CHROMOSOMAL MICROARRAY ANALYSIS
Chromosomal microarray analysis (CMA), also known as array-based comparative genomic hybridization (aCGH), is a technique to detect DNA copy number variations (CNVs) in the whole genome at a high resolution. In CMA, genomic DNA of the test sample is labeled with one fluorescent dye and control (reference) diploid DNA is labeled with another fluorescent dye (Figure 15-2). Equal amounts of the test and control DNA are cohybridized to a set of genomic fragments (oligonucleotide probes) spotted on a glass slide (array). The intensities of fluorescent dyes at each probe are measured by a scanner and compared between the test DNA and control DNA. If the intensities are equal at the given probe, the amount of the test DNA is interpreted as normal. Increased or decreased intensity for the test DNA over a control DNA indicates gain or loss in DNA amount respectively (Figure 15-2D, E). Most current CMA platforms contain from 60,000 to 400,000 oligonucleotide probes with comprehensive probe coverage within clinically relevant genes, as well as limited probes for the rest of the genome. CMA reliably detects DNA losses (deletions) and gains (duplications and triplications) as small as 200 kb in size, which gives at least 25-fold better resolution than G-banded chromosome analysis. The resolution of CMA depends on the total number of probes and average probe spacing across the genome. CMA can detect aneuploidy (monosomy and trisomy) and unbalanced structural rearrangements such as deletions, duplications, triplications, unbalanced insertions, isochromosomes, marker chromosomes, and complex chromosome abnormalities, but cannot detect balanced rearrangements or polyploidy. Arrays based on single nucleotide polymorphisms (SNP arrays), and the combination of oligonucleotide and SNP arrays, can detect the same abnormalities as CMA, as well as triploidies and copy number neutral chromosome abnormalities, such as contiguous regions of homozygosity that occur with uniparental isodisomy (UPD), consanguinity, or complete molar pregnancy. CMA, unlike G-banded karyotype, does not require cell culture, and can be completed within 3 to 5 days.