Genetics, Inborn Errors of Metabolism, and Newborn Screening

The Human Genome Project and the technological and information advances that it enabled mark the shift in clinical genetics from an observational and descriptive field to one of precise molecular diagnosis with a growing repertoire of meaningful therapeutic interventions. In the lifetime of most practicing senior neonatologists, that means going from a few recognizable syndromes, chromosome anomalies, and multifactorial birth defects to the more than 6000 phenotypes listed at the Online Mendelian Inheritance in Man (OMIM) website that have a “known” molecular basis. Using the search term “congenital” in OMIM identifies nearly 3600 listings.

Regardless of one’s experience, the volume of genetic information now available can be overwhelming. New tests and new testing methods seem to appear daily. New (and staggeringly expensive, in some cases) treatments for rare diseases emerge regularly. The reality is that the essential elements of good patient care have not changed, but more than ever, the practicing clinician needs a framework around which to organize their thinking about genetic problems. This chapter aims to accomplish that goal using an approach based on presentation in the NICU. It addresses birth defects, inborn errors of metabolism (IEM), newborn screening (NBS), and congenital neurologic abnormalities, providing an overall approach to diagnosis, critical elements of the initial evaluation and management, and when and how to involve other specialists. Finally, there is a brief discussion of newer methods of genetic testing that are revolutionizing care of the ill neonate.

Editorial comment:

Regarding the newer methods of genetic testing, “The rapid advancement of next-generation sequencing (NGS) technology and the decrease in costs for whole-exome sequencing (WES) and whole-genome sequencing (WGS), has prompted its clinical application in several fields of medicine. Currently, there are no specific guidelines for the use of NGS in the field of neonatal medicine and in the diagnosis of genetic diseases in critically ill newborn infants. As a consequence, NGS may be underused with reduced diagnostic success rate, or overused, with increased costs for the healthcare system.” ^a

a Borghesi A, Mencarelli MA, Memo L, et al. Intersociety policy statement on the use of whole-exome sequencing in the critically ill newborn infant. Ital J Pediatr. 2017;43:100;

Van Diemen ^b

b van Diemen CC, Kerstjens-Frederikse WS, Bergman KA, et al. Rapid targeted genomics in critically ill newborns. Pediatrics. 2017;140(4). e20162854.)

prospectively studied the speed and yield of rapid targeted genomic diagnostics in 23 critically ill children younger than 12 months where a quick diagnosis could not be made after routine clinical evaluation and diagnostics. Targeted analysis of 3426 known disease genes was performed by using WGS data. They measured diagnostic yield, turnaround times, and clinical consequences. A genetic diagnosis was obtained in 7 patients (30%), with a median turnaround time of 12 days (ranging from 5–23 days), including the identification of a new microdeletion in a child with a cardiomyopathy. They concluded that rapid targeted genomics combined with copy number variant detection adds important value in the neonatal and pediatric intensive care setting. We concur.

History and Physical Examination

Most of the information needed for genetic assessment is standard in the evaluation of the ill neonate. Pregnancy history, including careful documentation of exposures to medications and potential environmental toxins, is important, as is information about maternal illness that may impact embryonic development (e.g., diabetes mellitus, thyroid disease, or autoimmune disorders). Special attention should be paid to the history of pregnancy loss in the parents or family, other infants and children with health problems, and potential consanguinity (often associated with parents from a social structure with limited diversity or cultural norms that encourage marriage between cousins).

Physical examination is not different from standard approaches in the neonatal intensive care unit (NICU), although special attention should be paid to some aspects that may be overlooked during the initial assessment. Examples include evaluation of the umbilical cord looking for the typical three vessels; evaluation of the placenta for abnormalities or diagnostic clues; careful evaluation of the palate and uvula, looking for evidence of subtle manifestations of clefting (palpable defects of the hard palate with a submucosal cleft or bifid uvula), especially if the infant is to be intubated; evidence of skeletal malformation of extremities or chest, or even evaluating number and appearance of digits before tape is applied during intravenous (IV) placement; hypogonadism in females may be indicated by lack of the typical clitoral enlargement present in the newborn; skin pigmentation defects can be easily overlooked in the ill neonate, and should be specifically looked for as surrounding equipment and coverings are applied; and absence of typical posturing in the newborn as an indicator of subtle hypotonia.

Historically, there has been a perception that certain “dysmorphic” findings are critical. This can sometimes be misleading. “Low-set ears,” fifth finger clinodactyly, unilateral single transverse palmar crease, and increased “sandal gap” between the first and second toes are all examples of findings that may have some diagnostic significance but should not by themselves be drivers of further investigation. An infant with low muscle tone and “low-set ears” requires a careful genetic evaluation for the hypotonia, regardless of the facial appearance. It is rare that subtle “dysmorphic” findings in the absence of other more significant medical issues will lead to a diagnosis that changes patient management. There are certain findings that are critical in that they may indicate underlying defects not apparent superficially. Examples include very closely spaced eyes that can be associated with holoprosencephaly, abnormal chest shape or disproportion of extremities that may indicate an underlying skeletal abnormality, or patchy skin hyper- or hypopigmentation that may indicate a variety of genetic disorders.

Common laboratory tests performed in ill neonates may also carry clues to genetic abnormalities, particularly persistent abnormal findings that are not explained by other known diagnoses. Examples include persistent metabolic acidosis that may indicate a defect in metabolism of a specific organic acid (high anion gap) or underlying kidney disease (normal anion gap, hyperchloremic acidosis), persistent increases alanine aminotransferase and aspartate transaminase, which may indicate underlying liver or muscle disease, unexplained abnormal blood clotting parameters that could indicate liver disease or inherited coagulation defects, or persistent diffuse abnormalities of urine analytes that are reported normalized to urine creatinine that could indicate an underlying defect of creatine (and creatinine) synthesis.

Other clinical signs that suggest the possibility of an underlying genetic disorder are the involvement of multiple organ systems that are not easily explained by hypoxia or other findings. Any organ malformation, especially the brain, should raise the question of a genetic abnormality. Premature delivery or fetal distress during delivery that are not clearly related to recognizable causes should also raise the level of suspicion for an underlying genetic disorder. It is worth remembering that problems in labor and delivery with abnormal neurological status in the first days of life are common in infants eventually determined to have underlying genetic disorders; thus, difficulty in labor and delivery may be the first indication of an underlying genetic disorder, rather than the cause of later neurological findings.

Birth Defects and Multiple Congenital Anomalies

Major structural birth defects are discovered in 1 of every 33 births. Birth defects may occur as a result of maternal exposures or an abnormal uterine environment; however, the vast majority have an underlying genetic cause.

The first steps in the workup of any infant found to have a birth defect are a thorough physical examination to look for any other abnormalities that may be associated with the original finding and to define the phenotype through ancillary tests and imaging ( Box 6.1 ). Imaging studies to look for internal defects of the heart and kidneys should be performed and an ophthalmologist consulted to do a formal eye examination. Depending on the malformations present, further imaging and laboratory evaluation may be warranted.

BOX 6.1

Recommendations Following the Discovery of any Major Birth Defect

Physical Examination

Examine the head for shape and size, patency of the fontanelles, ridging of the sutures, presence of cutis aplasia or other scalp defects.
Examine the sclera of the eyes, assess for colobomas.
Examine palate (palpate with finger and also be sure to visualize the uvula—an abnormally shaped uvula may be indicative of a subtle soft palate defect).
Assess the extremities paying attention to limb lengths, bowing or abnormal alignment of the limbs, range of motion of the joints, number and shape of the digits, presence of syndactyly.
Examine the abdomen for the presence of large hernias, other abdominal wall defects, and hepatosplenomegaly.
Assess the genitalia for development of the external sex organs, ensure anal position and patency.
Examine the spine, looking for hair tufts or dimples indicative of a vertebral defect or cord tethering.
Examine the skin for appropriate pigmentation, hypo- or hyperpigmented lesions, bullae, abnormal scaling, abnormal hair growth.

Imaging/Procedures

Echocardiogram
Renal ultrasound
Hearing examination

Consultations

Ophthalmology for eye examination
As indicated by findings

Aneuploidies

Multiple, severe congenital anomalies in conjunction with or without an underlying heart defect should always raise suspicion for a chromosomal abnormality ( Table 6.1 ). Chromosomal microarray (CMA) is now the preferred method of assessing for gains and losses of genetic material (also referred to as copy number variants, or CNV). In the case that an aneuploidy is highly likely, evaluation by karyotype or fluorescence in situ hybridization may be sufficient to confirm the diagnosis. If CMA is performed, a karyotype can still be valuable and can be performed at the same time or in series to rule out a complex rearrangement or balanced translocation disrupting an important gene or genomic region.

TABLE 6.1

Cardiac Findings Frequently Associated With Specific Genetic Disorders

Syndrome	Associated Heart Defects (Most Common)	Gene(s)	Other Physical Findings
Turner syndrome (monosomy X)	Coarctation of the aorta Bicuspid aortic valve	NA	Short stature Low-set ears Short neck (webbed neck, low posterior hairline) Broad chest, widely spaced nipples Lymphedema Kidney malformations
Down syndrome (trisomy 21)	Endocardial cushion defects	NA	Epicanthal folds Upturned palpebral fissures Flat facial profile, flattened nasal bridge Small mouth, large tongue Hypotonia Single palmar creases, clinodactyly Space between first and second toes (sandal gap toes)
22q11 deletion syndrome	Conotruncal heart defects	NA	Low-set earsTubular nose (hypoplastic ala nasi)Cleft palate, submucosal cleft, bifid uvulaLaryngotracheoesophageal abnormalitiesKidney anomaliesHypocalcemiaThymus aplasia
Williams syndrome (deletion 7q11.23)	Supravalvular aortic stenosis Peripheral pulmonic stenosis	NA	Broad foreheadStellate pattern of the irisWide mouth with full lipsSmall chinHypercalcemia
CHARGE syndrome	Conotruncal defects AV canal defects Aortic arch defects ASD/VSD/PDA	CHD7	Colobomas Choanal atresia/stenosis Cranial nerve defects Hearing loss Abnormal external ears Cryptorchidism/hypogonadotropic hypogonadism Growth deficiency Cleft lip/palate Tracheoesophageal fistula
Noonan syndrome	Pulmonary valve stenosis ASD Hypertrophic cardiomyopathy	PTPN11 SOS1 RAF1 RIT1 KRAS NRAS BRAF MAP2K1	Low set ears Hypertelorism Down-slanting palpebral fissures Ptosis Broad/webbed neck Pectus Widely spaced nipples Cryptorchidism
Holt-Oram syndrome	ASD VSD Cardiac conduction defect	TBX5	Upper limb malformation (involving the carpal, redial, and thenar bones)
Alagille syndrome	Peripheral pulmonary artery stenosis Tetralogy of Fallot VSD ASD Aortic stenosis Coarctation of the aorta	JAG1 NOTCH2	Liver dysfunction Cholestasis Bile duct paucity Posterior embryotoxon Butterfly vertebrae Kidney abnormalities Pancreatic insufficiency Facial features: prominent forehead, pointed chin, deep-set eyes, hypertelorism, saddle or straight nose
Kabuki syndrome	Left-sided obstructive lesions Septal defects	KMT2D KDM6A	Elongated palpebral fissures with eversion of lateral lower eyelid Arched/broad eyebrows Short columella Large cupped ears Ear pits Cleft lip/palpate Blue sclerae Strabismus Ptosis Anorectal abnormalities Genitourinary abnormalities

ASD , Atrial septal defect; NA, not applicable; PDA, patent ductus arteriosus; VSD, ventricular septal defect.

FISH (fluorescence in situ hybridization) testing utilizes fluorescent probes that are designed to hybridize to specific regions of interest on the chromosomes. It can generally yield a result within 24 to 72 hours and is one of the fastest genetic testing modalities available. FISH can rule in or out a chromosomal aneuploidy with a high level of confidence. However, FISH testing will only reveal the presence or absence of the specific chromosome anomaly that is being assessed and will not yield additional findings of chromosomal duplications or deletions outside of those indicated in the test order. If a specific defect is likely, for example, trisomy 21, FISH is currently the fastest and most cost-effective test.

A karyotype will also determine the presence of aneuploidy with high confidence and would additionally provide information regarding the presence of large chromosomal duplications or deletions, as well as whether the aneuploidy resulted from a balanced translocation in the parent. Karyotypes generally take a longer time to result compared with FISH, which should be considered if making a diagnosis is time sensitive.

Microdeletions and Microduplications

22q11.2 Deletion Syndrome

The 22q11.2 deletion syndrome, previously referred to as DiGeorge syndrome or velo-cardio-facial syndrome before a common genetic etiology was identified, has a prevalence around 1 in 6000 births. The phenotypic spectrum of 22q11.2 deletions is quite variable, and occasionally a previously unrecognized parent will be discovered to carry the deletion after their child is born with a serious cyanotic heart lesion. Thus, any infant born with a conotruncal heart defect should be evaluated for the 22q11.2 deletion, even in the absence of other features of the syndrome, which include palatal or laryngotracheoesophageal abnormalities, low-set ears, hypoplastic alae nasae (nostrils), kidney anomalies, hypocalcemia, or an immune defect. Given the potential for serious adverse symptoms of hypocalcemia, if 22q11.2 deletion is suspected, calcium levels should be monitored while awaiting the genetic testing results.

Williams Syndrome

A diagnosis of Williams syndrome (7q11.23 deletion syndrome) may be difficult to make in the neonatal period based on physical features alone, as the unique facial appearance and personality typical of Williams syndrome may not become clearly apparent until later in childhood. The finding of supravalvular aortic stenosis or peripheral pulmonic stenosis should raise suspicion for the condition and lead to genetic testing. Additional findings of a broad forehead, stellate pattern of the iris, wide mouth with full lips, small chin, or hypercalcemia on laboratory testing would further support testing for Williams syndrome.

Testing for Microdeletions and Microduplications

Targeted FISH testing can be used for the evaluation of common microdeletion syndromes such as 22q11.2 and Williams syndrome (7q11.23 deletion). As with aneuploidy testing, FISH for a specified microdeletion can yield a result in 24 to 72 hours.

For isolated cardiac defects, or when multiple congenital anomalies are present but do not necessarily fit with a classically described syndrome, CMA is the recommended first-tier test. CMA offers a platform for broader testing for small chromosome microdeletions and duplications across the genome. A CMA can be used to test for both 22q11.2 deletion syndrome and Williams syndrome, and also identifies other aneuploidies and CNVs. The turnaround time for a CMA result is generally longer than FISH, ranging from 1 to 2 weeks. Thus, FISH testing offers a faster means to make a diagnosis when a particular chromosomal abnormality is suspected.

Given the large amount of normal variation in the genome across the population, there is always potential for a CMA to reveal a copy number variant of uncertain significance. These variants may represent pathogenic changes, or they could represent benign, normal variation. Testing of asymptomatic parents can usually help to clarify the significance of these changes, although this adds another step to the diagnostic workup. As these variants of uncertain significance can cloud and confuse the diagnostic picture, generally, if there is high suspicion for a particular condition such as 22q11.2 deletion syndrome, targeted testing through FISH is preferred over broader based testing, as this excludes the possibility for unclear variants to be discovered.

Congenital Heart Disease

Congenital heart malformations are a frequent finding in newborns, with an incidence of about 1% in the United States. Of these, about 25% have major structural defects leading to cyanotic disease that often requires early surgical or medical intervention. Any significant heart defect should raise the physician’s suspicion for a possible genetic abnormality and prompt a careful investigation to look for other clues that may point to a recognizable underlying syndrome. Associated findings of a cleft lip or palate, eye abnormalities, limb anomalies, kidney and genital defects, or a family history of cardiac malformations can help further guide the genetic evaluation by narrowing the differential diagnosis. A genetic etiology for isolated heart defects is often difficult to identify, given the broad differential diagnosis it encompasses. Many experts, although certainly not all, consider any major cardiac malformation, even in the absence of other syndromic features, as an indication for a basic genetic evaluation, specifically chromosome analysis.

The differential diagnosis for genetic causes of congenital heart disease is extensive, including both chromosomal abnormalities and single-gene defects. Among the chromosomal abnormalities, aneuploidy syndromes such as trisomy 21, trisomy 13, trisomy 18, and monosomy X often have major structural heart malformations. Microdeletion syndromes, including both the 22q11.2 deletion (velocardiofacial and DiGeorge syndrome) and 7q11.23 deletion (Williams syndrome), are also commonly associated with heart defects. Single-gene disorders to consider include CHARGE, Noonan, Holt-Oram, Alagille, and Kabuki syndromes. Heart malformations may also stem from nongenetic causes such as exposure to teratogens. The VACTERL spectrum featuring vertebral anomalies, anogenital defects, cardiac malformations, tracheoesophageal fistula, renal anomalies, and limb defects is not uncommon but should not be considered as a final diagnosis; rather, it is a phenotypic description. Children with VACTERL spectrum defects have been shown to occasionally have CNVs on CMA testing, and recently, overlap with defects of nicotinamide adenine dinucleotide (NAD+) synthesis was identified by genomic sequencing.

Establishing the genetic basis of a cardiac defect in the neonatal time period may not be practical in all circumstances. The diagnostic process may be prolonged, but ruling out the common genetic conditions early on is important, as a confirmed diagnosis will have significant implications for future care. For example, identifying a 22q11.2 deletion would prompt careful monitoring of calcium levels and investigation for an underlying immunodeficiency, both critical elements for management and for counseling the family before taking the child home. Identification of a patient with Alagille syndrome has major implications for monitoring and management of hepatic manifestations. Given the associated morbidity and shortened life expectancy seen with trisomies 13 and 18, diagnosis of either of these conditions may impact decisions to pursue major corrective heart surgery or how aggressive to be with medical therapies.

Evaluation: Many genetic syndromes have characteristic heart lesions, and the finding of a particular lesion should certainly lead to the inclusion of the associated syndrome in the differential diagnosis. The physician should always keep in mind that with most genetic conditions, a wide phenotypic spectrum of disease exists. Thus, the absence of the classic cardiac malformation does not exclude a syndrome from the differential. Table 6.1 lists some of the more common associations.

Following characterization of the underlying heart defect, further evaluation of a neonate with congenital heart disease should focus on identifying any other abnormalities that may help focus the genetic evaluation. These include:

ophthalmologic examination
thorough examination of the palate
renal ultrasound
examination of the genitalia and determination of patency of the anus
careful examination of the limbs and digits
X-rays of the spine

Single-Gene Disorders

There are numerous single-gene disorders that are associated with cardiac defects. As mentioned previously, some of the more commonly described single-gene syndromes shown in Table 6.1 include CHARGE syndrome, Noonan syndrome, Holt-Oram, Alagille syndrome, and Kabuki syndrome. Testing for these conditions involves sequencing of the genes in question. Consultation with a clinical geneticist who is familiar with ordering these tests is recommended when one of these conditions is being considered.

Orofacial Clefts

Orofacial clefts affect approximately 1 in 700 neonates. Cleft lip and/or palate (CLP) is a common feature of many syndromes; thus, once a cleft has been identified, as with other major birth defects, a thorough evaluation for other anomalies needs to take place.

Any child identified with a cleft palate should be evaluated for the Pierre Robin sequence. The Pierre Robin sequence is characterized by a U-shaped palatal cleft accompanied by micrognathia and backward displacement of the tongue (glossoptosis). This can have significant consequences for the airway and may even necessitate intubation or placement of a tracheostomy to ensure airway patency.

Syndromic Cleft Lip and/or Palate

Syndromic causes of CLP include (but are not limited to) Stickler, 22q11.2 deletion (discussed earlier), Treacher Collins, CHARGE, and Smith-Lemli-Opitz (SLO) syndromes.

Stickler Syndrome

Stickler syndrome is an important cause of cleft palate, often in the setting of the Pierre Robin sequence. Apart from morbidity associated with the airway, individuals with Stickler syndrome have high myopia with a significant risk for retinal detachment. Thus, evaluation and long-term follow-up by an ophthalmologist is critical, as is prompt treatment by ophthalmology should any change in vision occur. Stickler syndrome should be suspected in infants with significant myopia in the neonatal period, when hyperopia is the norm. Stickler syndrome is also associated with hearing loss, and thus, audiometry should be performed in the NICU and followed routinely. Stickler syndrome is a clinical diagnosis, but the genetic basis for Stickler syndrome has been established in recent years and includes mutations in one of six collagen genes. Molecular panel testing for these genes should be performed when Stickler syndrome is suspected.

Treacher Collins Syndrome

Treacher Collins syndrome (TCS) is a recognizable autosomal dominant condition caused by mutations in one of three genes: TCOF1 , POLR1C, or POLR1D . TCS is characterized by underdevelopment of the facial bones (namely the zygomatic arch and mandible) leading to midface hypoplasia and retrognathia. Choanal atresia and the Pierre Robin sequence can lead to significant airway issues necessitating a tracheostomy. External ear anomalies, hearing loss, colobomas of the lower eyelid, and other refractive errors of the eyes are also common. Cognition in TCS individuals is generally normal, and other organ systems, apart from the facial abnormalities, are usually unaffected.

CHARGE Syndrome

CHARGE syndrome features C oloboma, H eart defects, C hoanal atresia, R estricted growth and development, G enital anomalies, and E ar anomalies. Orofacial clefts are seen in approximately 15% to 20% of patients with CHARGE. Tracheoesophageal fistulas can sometimes be seen, as well as cranial nerve dysfunction, hearing loss, and swallowing problems. The only known gene associated with CHARGE is CHD7 , which is inherited in an autosomal dominant manner, usually attributed to a de novo mutation in the affected child. One-third of patients with a clinical diagnosis of CHARGE have no diagnostic genetic change in CHD7 , however, and the diagnosis is made clinically based on the presence of enough features of the condition to meet diagnostic criteria.

Smith-Lemli Opitz

Smith-Lemli Opitz (SLO) syndrome is a congenital disorder resulting from a defect in cholesterol biosynthesis (7-dehydrocholesterol reductase deficiency). A high-arched or cleft palate is a common feature of this disorder. Patients present with a variable degree of other features, which can include microcephaly, facial dysmorphisms (short nose with anteverted nares, broad nasal bridge, long philtrum, micro/retrognathia, blepharoptosis, low-set ears), 2-3 toe syndactyly (a hallmark sign of the condition), genital abnormalities, congenital heart defects, and abnormalities of the kidneys, adrenal glands, lungs, and intestinal tract. Patients may also present with a disorder of sex development (see later). Cholestatic jaundice and neonatal ascites can be observed, likely as a result of liver injury caused by accumulations of upstream products of the cholesterol pathway. Poor growth, intellectual disability, and behavior problems become apparent as the child grows older. Screening for SLO is accomplished by measuring serum 7,8-dehydrocholesterol levels. Significant elevations are indicative of disease. Low cholesterol levels are often observed as well, and there is a correlation with disease severity, magnitude of elevation in 7,8-dehydrocholesterol, and the degree of cholesterol deficiency.

Isolated Cleft Lip and/or Palate

It is not uncommon for CLP to be found as an isolated defect. Numerous studies have been performed in recent years to try and identify the etiology of isolated CLP, and mutations in numerous genes have been revealed. The most common genetic etiology for isolated CLP is Van der Woude syndrome, resulting from mutations in the gene IRF6 . However, it has become clear that most of the implicated genes, including IRF6 , have reduced penetrance, and there is a great deal of influence from environmental factors, as well as background genetics, in CLP. As the various genetic causes of isolated CLP have not been found to be associated with the development of any other major medical issues, identifying a genetic basis is typically not pursued, although, in light of the variable penetrance of mutations, a clinically unaffected parent could have an increased recurrence risk identified by molecular testing. It is likely that molecular testing will become more common with increasing knowledge of causative genetic defects and reduction in the cost of testing. Referral to a center experienced in multidisciplinary care of orofacial clefts will allow up-to-date counseling for the family.

The most important information to provide families of a newborn with isolated CLP is the risk for recurrence in future offspring. Generally, regardless of the genetic mutation, a 4% risk for recurrence of CLP can be quoted for any future sibling or offspring of the affected individual. In the case of a unilateral cleft lip without a cleft palate, or a cleft palate without a cleft lip, the recurrence risk to siblings or offspring falls to 2%. In the case of a bilateral cleft lip and palate, the recurrence risk is 6%. The risk further increases in when another affected sibling or offspring is born.

Disorders of Sex Development (Ambiguous Genitalia)

Disorders of sex development (DSD) often present with ambiguous genitalia, but can sometimes not manifest until the neonate displays signs of hypoglycemia, hyponatremia, or shock without appreciable abnormal physical signs. Other DSDs may not be apparent until later in life, such as when a phenotypic female fails to undergo menstruation and is found to have male internal anatomy. The etiologies of the different disorders are numerous and stem from defects in hormonal pathways, chromosomal disorders, as well as defects in genes involved in male patterning during development. Thus, workup for DSD warrants consultation with both endocrinology and genetics.

Ambiguous genitalia can lead to significant parental anxiety and strife. It is important not to label the infant with any particular gender until a genetic sex and etiology of the disordered sex development is determined. Providing the family with resources for psychosocial support in dealing with the uncertainty may be necessary. Furthermore, consultation with urology and surgery may also be warranted for surgical correction of the genitalia once a decision has been made as to which sex the baby will be raised.

For any infant presenting with ambiguous genitalia, the priority is to ensure that they are medically stable with normal blood pressure, heart rate, serum sodium concentrations, and glucose levels. Any abnormalities of these parameters should prompt medical intervention. Once the neonate is appropriately stabilized, the next step is to determine the genetic sex of the baby. This involves sending a karyotype or FISH testing for the sex chromosomes. Knowing the genetic sex of the baby allows the physician to determine if the baby is over- or undervirilized and thus guide further workup. Roughly 80% of all neonates with ambiguous genitalia have an XY karyotype, with 10% to 15% carrying an XX genotype and the rest demonstrating sex chromosome abnormalities.

Abnormalities in blood pressure, hyponatremia, or hypoglycemia, especially in the setting of ambiguous genitalia, should raise concern for adrenal insufficiency. Testing considerations include a serum cortisol, 17-hydroxyprogesterone, testosterone, and growth hormone. Further recommendations may be added by endocrinology. In all cases of ambiguous genitalia, testosterone levels should be measured, even in the absence of blood pressure or chemistry abnormalities. Abnormalities in hormone levels should be addressed by an endocrinologist and treated appropriately. Further investigation as to the etiology of these abnormalities may be warranted as addressed later.

Careful examination of the genitalia should be performed, paying attention to presence of palpable testes, scrotal/labial fusion, phallic/clitoral size, and presence of a urogenital slit/hypospadias. Furthermore, imaging of the internal anatomy to characterize the reproductive organs should be performed. Attention should also be paid to other minor anomalies on physical exam that may indicate a specific disorder of sex development. Hypotonia and feeding issues, in conjunction with hypoplasia of the genitalia in a male, may indicate Prader-Willi syndrome (PWS). Dysmorphic facies, 2-3 toe syndactyly, and undervirilized male genitalia would be suspicious for SLO syndrome. Testicular tissue in a female should be removed to reduce risk of later malignancy.

Congenital Adrenal Hyperplasia

The most common cause of congenital adrenal hyperplasia is 21-hydroxylase deficiency. In this disorder, there is a block in the pathway for aldosterone and cortisol synthesis. Thus, the infant presents with adrenal insufficiency that can manifest with shock-like symptoms of hypotension, hyponatremia, and hypoglycemia. In addition to lack of cortisol and mineralocorticoids, metabolites before the enzymatic block accumulate and are shunted toward the sex-steroid pathway with a consequent increase in androgen production. Increased androgens lead to virilization in females. Genotypic males may not have any apparent genital anomalies but will present similar to females with adrenal insufficiency and salt wasting. Cortisol levels will be low in these patients, and elevations in 17-hydroxyprogesterone are observed.

Other defects in the adrenal-steroid hormone synthesis pathway that can lead to disordered sex development include 11-beta-hydroxylase deficiency, 17-alpha-hydroxylase deficiency, and 3-beta-hydroxysteroid dehydrogenase deficiency. In 11-beta-hydroxylase deficiency, the enzymatic block is distal to that of 21-hydroxylase, and although aldosterone and cortisol production is impaired, there is enough activity in the metabolites before the block to prevent adrenal crisis. However, as the regulatory axis is impaired, the precursors of 11-beta-hydroxylase deficiency accumulate and can be shunted toward androgen synthesis and cause female virilization as well as salt retention and consequent hypertension from the accumulation of mineralocorticoids. 17-alpha-hydroxylase deficiency results in a block in sex hormone synthesis and leads to ambiguous genitalia in XY individuals and failure of pubertal development in all affected individuals. 3-beta-hydroxysteroid dehydrogenase deficiency causes a block in the synthesis of cortisol, aldosterone, and sex-steroid hormones and thus will lead to adrenal crisis in addition to undervirilization in males, with lack of puberty in both sexes. As cholesterol is the precursor for all steroid hormone synthesis, defects affecting cholesterol synthesis and transport can also lead to adrenal insufficiency and virilization issues.

In all cases of congenital adrenal hyperplasia, a definitive diagnosis with molecular testing of the suspected genetic defect should be pursued.

5-Alpha-Reductase Deficiency

Individuals with 5-alpha-reductase deficiency lack the enzyme needed to convert testosterone to dihydrotestosterone (DHT). DHT is a potent androgen for signaling the formation of the external male genitalia, and as a result, males with 5-alpha-reductase deficiency are born with ambiguous or female-appearing external genitalia. As testosterone is still produced, the internal anatomy remains male, and these individuals will still develop and display secondary sex characteristics (although often to a lesser degree than their male peers). Basal levels of serum testosterone and DHT are not reliable for the diagnosis of 5-alpha-reductase deficiency, and stimulation testing with human chorionic gonadotropin is needed to clearly assess whether the levels are abnormal. Molecular testing of the SRD5A2 gene is generally the best initial test if 5-alpha-reductase deficiency is suspected.

Androgen Insensitivity

Androgen insensitivity syndrome, an X-linked disorder, manifests as a result of complete or partial dysfunction of the androgen receptor. Without normal androgen receptor activity, androgen hormones are unable to exert an effect (or have only a partial effect) on development of the male external genitalia. Thus, males are born with female external genitalia. Imaging studies will reveal an absent uterus and the presence of testes. The vagina often ends in a blind pouch. Unless a karyotype is performed, the diagnosis is often missed, and the baby will be considered a normal female. Other clues in the newborn period for this diagnosis are palpable testes in the inguinal area or an inguinal hernia. Many patients will not be diagnosed until the teenage years when they present for evaluation of primary amenorrhea. In the neonatal period, the diagnosis is generally made through molecular testing of the AR gene.

Exogenous Testosterone

A careful history should be taken for any baby born with ambiguous genitalia, but especially in any genotypic females presenting with virilization. Exogenous testosterone use by the father (or mother) during the pregnancy, which may have come in contact with the mother’s skin and been absorbed into her circulation, can lead to virilization in the female neonate in the absence of any underlying genetic defect.

Non-endocrine Causes of Disordered Sex Development

When no steroid/hormonal or other phenotypic anomalies are consistent with a specific diagnosis, the next step in the workup of the infant with ambiguous genitalia is to look for genetic changes that may have affected sex differentiation in utero. The first place to start is with chromosome evaluation. The CMA will be able to identify any duplications or deletions of the chromosomes in regions containing genes known to be involved in sex differentiation. These genes include those in the SRY region on the Y chromosome, the DMRT1 on 9p, and several others. If no chromosomal changes are evident, further investigation with molecular testing, under the guidance of a clinical geneticist or another team experienced in genetic evaluation of DSD, is warranted.

There are multiple chromosomal and single-gene causes of ambiguous genitalia. Discussed are a few of the more common diagnoses to consider, although this list is by no means exhaustive.

WAGR: WAGR syndrome (Wilms tumor, aniridia, genital anomalies, and mental retardation) is a contiguous gene deletion syndrome occurring on chromosome 11p13. The genes in the region include WT1 predisposing to Wilms tumor and PAX6 leading to the aniridia. The other features of the syndrome are believed to be secondary to other affected genes in the region. Genital abnormalities can include cryptorchidism (most common), hypospadias, as well uterine and ovarian abnormalities. Ambiguous genitalia can be seen.

SLO : SLO syndrome is a congenital disorder resulting from a defect in cholesterol biosynthesis described previously.

Abdominal Wall Defects

The most common abdominal wall defect identified in the neonate is an umbilical hernia. Most umbilical hernias are small, freely reducible, and do not require any intervention. General guidelines are to repair the defect only if it has not spontaneously closed by 5 years of age. Earlier repair may be required if there are concerns that the hernia is growing in size, is larger than 1.5 cm, or if there are concerns for strangulation of the intestine. In the absence of other findings, genetic workup for an umbilical hernia is not necessary. However, large umbilical hernias can, in some cases, represent an attenuated form of an omphalocele. Thus, if a large umbilical hernia is found in conjunction with other birth anomalies, genetic evaluation (similar to that of an omphalocele, discussed later) should be pursued.

The two most common major abdominal wall defects encountered in the NICU include gastroschisis and omphalocele. The largest distinction between the two is the presence of a peritoneal sac enclosing the abdominal organs extruding through the umbilical ring in an omphalocele, whereas in gastroschisis, the abdominal organs spill out of the abdominal defect without a covering membrane, typically beside the umbilical ring.

Gastroschisis is believed to stem from a vascular insult to the abdominal wall as it is forming. This insult results in failure of the abdomen to fully close, and the opening allows the abdominal organs to extrude from the abdomen during development. The defect is most often to the right of the umbilicus. The finding of gastroschisis (usually identified on prenatal ultrasound) requires careful planning in terms of delivery. It is important that the bowels are wrapped and protected in a plastic covering as soon as the baby is born to protect from water loss and thermal instability. Prompt evaluation and management by a pediatric surgeon is required.

At this time, an identifiable genetic cause for gastroschisis has not been found. The majority (∼85%) of these infants do not present with other birth defects, and their prognosis is good in terms of developmental and neurologic outcomes, pending appropriate surgical correction of the abdominal defect without significant complications. Syndromic causes are identified in approximately 15%, typically with other findings or malformations. Genetic testing, specifically CMA, should be considered in most cases, and certainly if there is any other malformation.

Omphaloceles are much more commonly associated with genetic changes; thus, genetic evaluation is always warranted. These babies, too, need prompt surgical attention following birth; however, given that they already have a sac covering their abdominal contents, the risks for infection and water loss are somewhat less. The abdominal contents protrude through the umbilicus with omphaloceles.

Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS) should be considered in any baby born with an omphalocele. These babies are generally large for age, can have omphalocele or large umbilical hernia, visceromegaly, large tongue, hemihypertrophy, and they are at risk for hypoglycemia in the newborn period. Given the associated risks for seizures and other morbidities associated with hypoglycemia, careful monitoring of blood sugars is warranted in the newborn period for any baby with an omphalocele or other findings concerning for BWS. BWS results from methylation defects at chromosome 11p15. Therefore, testing for BWS requires targeted methylation studies. Less commonly, maternally inherited mutations in the gene CDKN1C can cause BWS. If there is strong suspicion for BWS and methylation testing returns normal, sequencing of CDKN1C would be the next step in evaluation.

Aneuploidy

Trisomy 13 also is frequently associated with an inguinal or umbilical hernia or omphalocele. Findings of an omphalocele in conjunction with cutis aplasia, cleft lip, holoprosencephaly, heart defects, polydactyly, or other major malformations warrants an evaluation with FISH for aneuploidies or karyotyping to evaluate for trisomy 13 as well as trisomy 18, which can also be associated with omphalocele, although less commonly.

Other Single-Gene Disorders

Other considerations for a patient with an omphalocele include Donnai-Barrow syndrome, which features craniofacial dysmorphisms, eye abnormalities, hearing loss, and intellectual disability. Donnai-Barrow syndrome is caused by mutations in LRP2 gene. Fibrochondrogenesis is a severe short-limbed skeletal dysplasia and also often features an omphalocele. Fibrochondrogenesis is caused by biallelic mutations in the COL11A1 gene. Manitoba oculotrichoanal syndrome is caused by biallelic FREM1 mutations and is notable for dysmorphic craniofacial features of the hairline, eyes, and nose, and imperforate anus in addition to omphalocele or large umbilical hernia. In the absence of a recognizable syndrome, CMA testing, looking for small chromosomal deletions or duplications, should be performed.

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