This detailed physical exam assesses for defects in the structure or morphology of an organ or organ system that reflects an abnormal developmental process. In assessing for congenital defects and in order to develop an appropriate differential diagnosis, a detailed physical exam with emphasis on the craniofacial exam is performed and measurements are compared to published age-matched standards [4]. The initial assessment begins with the newborn growth parameters, adjusted for any prematurity. The height, weight, and head circumference will provide a snapshot view of fetal health and intrauterine environment. For example, head-sparing intrauterine growth retardation where HC >length >weight may reflect poor nutrition or placental insufficiency, while microcephaly might instead suggest underlying neurological malformation with a genetic etiology or infectious agent such as cytomegalovirus. The dysmorphology exam focuses on assessing the patient for anatomic features that diverge from normal standards with the understanding that some might be familial variants. The shape and size of the head and fontanelles and assessment of the cranial sutures should be obtained. The presence of craniosynostosis may significantly narrow down the diagnoses under consideration and an unusual head shape or size would prompt imaging studies that might reveal a brain malformation (Fig. 2a, b). In further assessing the head, notation of any scalp defects, differences in the shape of the forehead, eyebrows, and distribution of hair should be sought. The spacing of the eyes with measurements of the canthi and palpebral fissure lengths is performed. The presence of colobomata, cataracts, and the surrounding structures such as the presence of epicanthal folds or downward or upward slant of the palpebral fissures is also documented as these can be characteristic findings of some syndromes. Careful examination of the ears, assessing their placement, rotation and appropriate folding of the ear is important as is assessment for auricular pits and tags which can be specific for certain disorders. The nose is evaluated for proper formation of the bridge, alar nasi, nares, and nasal tip. Patency of the chonae is also assessed. The mouth and throat are evaluated for evidence of defects or clefts of the lip and/or palate. The shape of the palate and the uvula are examined, with direct implications for functional issues of the airway. The mouth is carefully examined for findings such as natal teeth, gum abnormalities, lip pits, frenulae, and tongue malformations that might be suggestive of a diagnosis. Evaluation of the chin is critical in a patient with airway involvement and micrognathia and retrognathia can be part of distinct syndromic entities. They can also occur as isolated malformations. Finally, the neck is evaluated for webbing, excess nuchal skin, or bony cervical abnormalities, which would prompt a subsequent cervical spine stability evaluation, affecting airway management.

Examination of the chest and thorax notes any bony deformities such as pectus or narrow chest and internipple distances. Abnormalities should prompt imaging studies such as chest X-ray evaluating for bone malformations, while presence of a murmur would prompt cardiac evaluation. The abdominal exam assesses primarily for evidence of abdominal wall defects such as omphalocele and for the presence of organomegaly, which is more suggestive of an inborn error of metabolism. When examining the umbilical cord, a 2-vessel cord with only a single umbilical artery may reflect the presence of a renal anomaly. The genitourinary exam assesses external genitalia where anomalies may be associated with internal malformations of the GU tract as well. Patency of the anus and its placement are assessed as this finding may present as part of a recognizable constellation of findings in a syndrome. The extremities are carefully examined for structural integrity of the digits, their formation and placement and palmar creases. Often, limb anomalies particularly those of the hands and feet can be a vital clue to an underlying diagnosis.

A dermatologic evaluation to examine for the presence of phakomatoses or skin manifestations that signal an underlying genetic diagnosis is important. For example, multiple café au lait spots or neurofibromas can be associated with neurofibromatosis type I. The irregular swirly pigmentation encountered with Hypomelanosis of Ito may be the only clue to an underlying chromosomal mosaicism. Here, the skin pigmentation patterns can represent the distinct chromosomal admixture. Hemangiomata are also noteworthy, particularly those of the face and neck as they may signal the presence of other such vascular malformations affecting the airway.

The neurologic exam, while clearly helpful for diagnostic purposes may have added impact on an infant’s ultimate prognosis. Evaluation of tone, basic reflexes, involuntary movements, and seizure activity are critical to management of the patient [5].

The field of genetics has experienced tremendous technological advances since the completion of the Human Genome Project in 2000. A brief review of techniques currently used in molecular diagnostics and their applications is discussed.

Methodology for identification and microscopic examination of the integrity of chromosomes became routinely available with the development of G-banding techniques in the 1960s. This staining methodology is still used today to identify aneuploidy involving whole chromosomes, or large (>5–7 Mb) rearrangements such as duplications and deletions within chromosomes. Standard G-banded karyotypes are typically performed on peripheral blood from the patient after stimulating the lymphocytes to grow and divide in culture. Usually, after 72 h in culture, a sufficient number of cells can be harvested to perform karyotyping. The process allows the cells to be in the metaphase stage of cell division when the chromosomes lengthen into distinct structures. Special Giemsa-based staining then allows the dark and light colored banding patterns to be discerned by further analysis by a cytogeneticist. This has been the standard technique used for detection and confirmation of common trisomies such as Trisomy 13, 18, or 21 or disorders of the sex chromosomes. Currently, it is still the only clinical diagnostic technique that can detect balanced translocations where chromosomal material from one chromosome is rearranged onto another. A balanced translocation does not result in a change in the dosage or copy number of a chromosomal region or of the genes residing in that region.

It is important to recognize that a normal karyotype (either from a prenatal CVS or amniocentesis specimen or from a postnatal blood sample) does not exclude the possibility of a chromosomal disorder. Due to the advent and rapid adoption of newer microarray technology described below, a large number of microdeletion/microduplication syndromes have been recognized, many of which are associated with congenital airway malformations. For example, the 22q11.2 microdeletion syndrome has been associated with cleft palate and laryngeal webs, which may lead to significant airway compromise during the neonatal period. Such submicroscopic gains and losses of chromosomal regions are considered copy number variations (CNVs). They may be detected within a chromosome (interstitial) or at the ends of the chromosome (subtelomere) and can result in change in dosage of a gene or genes located in a given region.

Fluorescence in situ hybridization or FISH is a molecular cytogenetic technique where a specific fluorescent-tagged DNA probe representing a single locus or part of a particular chromosomal region hybridizes to that region of the genome. It is useful in cases where the patient’s features are suggestive of a chromosomal disorder that is not typically visible under the microscope. Indeed, these submicroscopic genomic disorders have been, until recently, primarily identified by FISH and are associated with known clinical syndromes. The most frequently occurring microdeletion syndrome is the chromosome 22q11.2 deletion syndrome, associated with the clinical DiGeorge and Velocardiofacial (VCFS) syndromes. In addition, other chromosome-specific deletion syndromes such as those of chromosome 15q11.2 (Prader-Willi Syndrome and Angelman syndrome), chromosome 7q11.2 (Williams syndrome) have historically all been identified using a FISH probe for their respective regions. The disadvantage of this technique is that the clinician must know and specifically order the proper FISH test for a given chromosomal locus and many of the syndromes, even the well-recognized ones, can have atypical presentations or overlap with other diagnoses. In addition, in some cases, a patient may have a rearrangement that does not have the typical breakpoints associated with a given region and may not be detectable using the standard FISH probe. For these reasons, FISH has largely been replaced by chromosomal microarray analysis except for specific applications such as for testing family members of a proband.

FISH continues to be utilized for prenatal genetic testing. This modality can also be used to rapidly assess for trisomy or absence of whole chromosomes even without culturing and stimulating the cells toward metaphase. Such interphase FISH is routinely used as a rapid screen for aneuploidy of chromosomes 13, 18, 21, X and Y on chorionic villus sampling (CVS), amniocentesis, and in postnatal cases where rapid diagnosis may be desired for medical decision making, such as for an infant with features of Trisomy 13 or 18.

Over the last decade, single-locus testing by FISH has given way to chromosomal microarrays. This technique was originally based in comparative genome hybridization where patient DNA is labeled with one fluorescent probe while reference DNA is labeled in another and hybridized together. This allowed detection of regions across the genome, which had changes in copy number relative to the reference DNA and reflecting either gains (duplications) or losses (deletions) in the patient DNA. More recently, this approach has given way to the use of synthesized oligonucleotide probes or single nucleotide polymorphism (SNP)-based probes densely arranged on a microarray chip. Today, over two million such probes are routinely used on clinical diagnostic platforms for genome-wide postnatal testing. This allows the potential to report copy number changes in the 50–100 kb range. The processing is automated and copy number variation is compared against a large panel of standards. As might be expected with such high resolution analysis, changes that are of unknown significance are encountered and require further investigation and often examination of parental samples to help interpret the proband’s results.

Chromosome microarrays have provided a rapid, highly reproducible way to assess for submicroscopic chromosome rearrangements that might underlie an airway malformation, in an objective manner across the genome. It tests the regions associated with known syndromes and those not yet described, by identifying regions and groups of genes whose dosage has been changed due to the rearrangement. The widespread use of this technique for postnatal cases has led to the identification of several new genomic disorders. For these reasons, chromosome microarray has been recommended as the first-line study for cases of multiple congenital malformations and unexplained intellectual disability [6]. This recommendation has also been extended to prenatal cases of multiple congenital malformations [7].

The array techniques rely on the detection of copy number change relative to standards from arrays run on control individuals. Structural changes that do not result in copy number variation, such as a balanced translocation or inversion of part of a chromosome, will not be detected by this technique. For example, the presence of an extra copy of material from chromosome 21 will be detected on a microarray for a patient presenting with Down syndrome. However, whether this has occurred due to a trisomy (a free-standing extra chromosome 21) or from a translocation that might also occur in a balanced form and be carried by a healthy parent will not be differentiated by the microarray. Knowledge of the mechanism is necessary to provide an accurate recurrence risk for the parents and further testing and genetic counseling is indicated.

Single Gene Disorders. Many syndromes are due to the disruption of the normal function of a single gene or single gene disorder. An alteration of a single base pair in the sequence of the gene leads to a disruption in the gene’s functional product. In other cases, insertions or deletions of even a small number of nucleotides can disrupt the gene and subsequently, its protein product. These insertions or deletions may be as small as 2–3 base pairs, or may involve hundreds of base pairs. Neither a standard karyotype nor chromosomal microarray will detect these small sequence changes. These techniques may fail to detect deletions that encompass an entire gene, especially for small genes. Direct sequencing of the target gene will reveal single base pair mutations or small insertions and deletions. Larger deletions or duplications that involve either part or all of the gene are often even more difficult to detect, being too small to detect on a chromosomal microarray, but too large to detect with standard sequencing. These genetic lesions often require PCR-based deletion/duplication testing targeted to a specific gene locus. It is important to consider these potential mutational mechanisms and ensure they have been appropriately tested prior to eliminating a diagnosis from consideration.

Airway malformations can be part of a constellation of findings that result from mutation in a single gene. CHARGE syndrome is a good example, with mutations in the CHD7 gene associated with the clinical findings of CHARGE. Assessment of single gene disorders requires different techniques than those described above. Until recently, PCR-amplification and Sanger sequencing were the most commonly used techniques, requiring a gene-by-gene approach. These techniques are designed to examine the DNA at an individual nucleotide level assessing for alterations of one nucleotide for another (e.g., cytosine to adenine, C to A), deletion of one or more individual nucleotides or the insertion of a single or group of nucleotides into the normal sequence. The deletions or insertions have the potential to change the reading frame, resulting in a frameshift, which can affect production of the resulting mRNA and protein. A mutation that substitutes one nucleotide for another can result in formation of a premature termination codon and truncation of the putative protein. The mutation then is known as a nonsense mutation. An alteration in the DNA sequence that results in substitution of one amino acid for another in the translated protein, is known as a missense mutation, and can lead to production of a defective protein product. Gene sequencing can detect a variety of disease-causing mutations but requires that the clinician has selected the correct gene to test, which can be difficult in disorders with variable features.

Sequencing is not the testing method of choice for alterations where large segments of a gene are altered. For example, sequencing may miss cases where an entire exon of a gene is missing and not amplify properly to be sequenced. The exon deletion may be too small for accurate detection on a chromosome microarray. In these situations, specialized techniques involving quantitative PCR or MLPA may be used.

Standard PCR or polymerase chain reaction is commonly used to amplify desired segments of DNA; however, it is not typically quantitative or related to the amount of input DNA. Real-time PCR instead is a kinetic measurement, which assesses amplification in the early linear stages of the reaction and utilizes fluorescent labeling to quantitate products. The higher the copy number of a segment of DNA at the start, the more rapidly a significant increase in fluorescent product can be detected. MLPA or multiplex ligation-dependent probe amplification allows the relative quantitation of dozens of different PCR products of various lengths simultaneously. These two quantitative techniques allow for rapid assessment of copy number of a gene, part of a gene or a group of genes in a specific chromosome region. These diagnostic approaches require the selection of the appropriate target regions and unlike arrays, are not genome-wide. They are however, useful to test for intragenic deletions and duplications that might be below the threshold of reliable detection for microarray-based diagnostic testing and are routinely used in this capacity.