Pediatric Clinical Genetics

Andrew J. Green and James J. O’Byrne

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Diagnosis of Malformation Syndromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Definitions Used with Congenital Anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3A Clinical Genetic Approach to Diagnosis of Malformation Syndromes . . . . . . . . . . . . . . . 3

Genetic Etiology of Congenital Anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Chromosome Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Single-Gene Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Polygenic/Multifactorial Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Genetic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Chromosomal G-Banding Analysis (Standard Karyotyping) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Fluorescent In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Array Comparative Genomic Hybridization/

Chromosomal Microarray Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Specific Testing for Common Known Single-Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . 15Next-Generation Sequencing and Gene Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

AbstractThe primary aim of this chapter is to introducethe pediatric surgeon to the fundamental con-cepts of clinical genetics and equip him/herwith the basic genetic terminology and toolsto manage some of the malformation syn-dromes commonly encountered in surgical

A. J. Green (*)Department of Clinical Genetics, Our Lady’s Hospital,Crumlin, Dublin, Ireland

School of Medicine and Medical Science, UniversityCollege Dublin, Belfield, Dublin, Irelande-mail: andrew.green@olchc.ie; andrew.green@ucd.ie

J. J. O’ByrneDepartment of Clinical Genetics, Our Lady’s Hospital,Crumlin, Dublin, Irelande-mail: James.O'Byrne@olchc.ie; obyrnej@tcd.ie

# Springer-Verlag GmbH Germany, part of Springer Nature 2019P. Puri (ed.), Pediatric Surgery,https://doi.org/10.1007/978-3-642-38482-0_10-2

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practice, particularly during the newbornperiod. A clinical genetic approach to diagno-sis, etiology, and inheritance patterns of mal-formation syndromes are outlined. Geneticinvestigations, often employed to help uneartha diagnosis including chromosomal G-bandinganalysis/standard karyotyping, fluorescent insitu hybridization, and array comparativegenomic hybridization analysis/chromosomalmicroarray are described and discussed indetail along with single-gene tests and thedevelopment of next-generation sequencingand gene panels.

KeywordsChromosome disorders · Single-genedisorders · Karyotype · Microarray · Next-generation sequencing

Introduction

Genetic disorders are common in pediatric prac-tice and contribute to between 30% and 70% ofchildhood hospital admissions. Genetic condi-tions cause a range of clinical problems includingmalformations, metabolic disorders, learning dis-ability, or neurological disease. With over 5,000different genetic conditions, almost every aspectof pediatrics will involve managing children withgenetic disorders. Clinical geneticists can offerexpertise both in diagnosing rare and ultra-raregenetic disorders and advising families withknown genetic disorders. This chapter will focuson the approach a clinical geneticist takes whenreviewing infants and children with congenitalanomalies and diagnosing malformation syn-dromes, the different forms of genetic diseaseswhich can cause congenital anomalies, and theup-to-date genetic investigations that are availableto aid the clinician to arrive at these diagnoses. Itis hoped the chapter will equip the pediatric andneonatal surgeon with the baseline genetic knowl-edge required to manage those cases of childrenwith malformations who have (or may have) anunderlying genetic diagnosis.

Diagnosis of Malformation Syndromes

Introduction

One child in 40 (2.5%) is born with a significantmajor congenital anomaly which accounts for20–25% of perinatal and childhood mortality.Most affected children have a single isolatedmajor congenital anomaly, in the absence of anyunderlying syndrome. However, where a child hasmore than one congenital anomaly, with or withoutdysmorphic features, the possibility of an underly-ing genetic syndrome or association should be con-sidered. Awareness of this possibility is veryimportant for management of the patient and foradvising the whole family. Examples of the com-moner congenital anomalies, grouped by the organsystem affected, and the approximate birth inci-dence are shown in Table 1. It is also important tonote that approximately 10% of the normal popula-tion will have a minor congenital anomaly, such asfifth finger clinodactyly or a single palmar crease,

Table 1 Examples of major congenital anomalies

Examples of majorcongenital anomalies

Birth incidence (per1,000 births)

Cardiovascular 10

Ventricular septal defect 2.5

Atrial septal defect 1

Patent ductus arteriosus 1

Fallot’s tetralogy 1

Central nervous system 10

Anencephaly 1

Hydrocephalus 1

Microcephaly 1

Lumbosacral spina bifida 2

Gastrointestinal 4

Cleft lip/palate 1.5

Diaphragmatic hernia 0.5

Esophageal atresia 0.3

Imperforate anus 0.2

Limb 2

Transverse amputation 0.2

Urogenital 4

Bilateral renal agenesis 2

Polycystic kidneys(infantile)

0.02

Bladder extrophy 0.03

2 A. J. Green and J. J. O’Byrne

which, in the absence of any other problems, is ofno major significance.

Definitions Used with CongenitalAnomalies

Distinctions can be drawn between several differ-ent forms of congenital anomaly.

A disruption can be defined as an anomalywhich is caused by an interference in the structureof a normally developing organ. A good examplewould be the digital constrictions and amputationscaused by amniotic bands. Amniotic bands arestrands of tissue which cross from one wall ofthe amniotic sac to the other and can constrictparts of the developing fetus.

A deformation can be defined as an anomalywhich is caused by an external interference in thestructure of a normally developing organ. Anexample would be talipes equinovarus caused bychronic oligohydramnios, perhaps from anamniotic leak.

A malformation can be defined as an anomalywhich is caused by an intrinsic failure in thenormal development of an organ. Common exam-ples would be congenital heart disease, cleft lipand palate, and neural tube defects.

A dysplasia is an abnormal organization ofcells in a tissue, often specific to a particulartissue. For example, achondroplasia is a skeletaldysplasia caused by a mutation in the FGFR3gene. Most dysplasias are single-gene disorders.

A sequence can be defined as a primary mal-formation which results in secondary deforma-tions. An example is Potter’s sequence which isthe group of anomalies consisting of pulmonaryhypoplasia, oligohydramnios, talipes, cleft palate,and hypertelorism (see Fig. 1). All of these anom-alies arise as a result of the failure of urine pro-duction in the fetus. The cause of Potter’ssequence and failure of urine production couldbe posterior urethral valves, dysplastic or cystickidneys, or renal agenesis, all of which can havegenetic and nongenetic origins. Pierre Robinsequence is the grouping of cleft palate, micro-gnathia, and glossoptosis, which can have at least

30 different causes. A sequence therefore does nothave a specific cause or inheritance pattern.

An association can be defined as a clusteringof anomalies, which is not a sequence, whichoccur more frequently than by chance, but hasno prior assumption about causation. An exampleis the association of VATER, whose acronym ismade up from the grouping of vertebral anoma-lies, anal abnormalities, tracheooesophageal fis-tula, and radial or renal anomalies. There is noclear cause for VATER, although it can rarelyoccur in people with chromosome 22q11 micro-deletions and can also rarely be mimicked byFanconi’s anemia.

A syndrome is a description of a group ofsymptoms and signs and a pattern of anomalies,where there is often a known cause, or an assump-tion about causation. The looser definition of“syndrome” to describe any anomaly should beavoided. The term can include chromosomal dis-orders such as Down’s syndrome or single-genedisorders such as van der Woude syndrome whichcan cause cleft lip and palate with lower lip pits.

A Clinical Genetic Approachto Diagnosis of MalformationSyndromes

History: When reviewing a child with a congeni-tal anomaly, a comprehensive medical history andexamination is an essential starting point toward adiagnosis. Several important aspects of the historyneed to be explored including a detailed three-generation family history, with reference notonly to a history of the same anomaly, but otheranomalies as well. The history should includedocumentation of consanguinity, ethnicity, preg-nancy losses, stillbirths, and neonatal deaths andany history of potential teratogens in the preg-nancy, considering the likely embryologicaltiming of the anomaly. Teratogens can includeprescribed medications that the mother has takenduring pregnancy, recreational drugs, maternaldiabetes mellitus, and prolonged maternalhyperthermia.

Examination and Investigations: In the pres-ence of one congenital anomaly, a very careful

Pediatric Clinical Genetics 3

examination should be carried out to check for anyother subtle abnormalities or for dysmorphicfacial features, e.g., to check for hydrocephalusin an infant with a spinal meningomyelocele. Adiagnostic approach to congenital anomalies isoutlined in Fig. 2. Deformations and disruptionsneed to be excluded first. If the pattern ofmalformations fits into a well-described malfor-mation sequence, then a cause for that sequenceshould be sought. If the anomalies do not fit into asequence, then a syndrome or association diagno-sis should be attempted. If a malformation syn-drome diagnosis is achieved, it is important toremember that syndromes can be caused by chro-mosomal, single-gene (monogenic), multiple-gene (polygenic) disorders or by environmentalagents. If cause is unknown and there is more thanone malformation or significant dysmorphology,

chromosomal analysis should be requested. Aclinical genetic opinion should also be sought, asa clinical geneticist can often help greatly inachieving a diagnosis, as well as in counselingparents about the likelihood of recurrence of sim-ilar problems in other family members.

Genetic Etiology of CongenitalAnomalies

Introduction

The genetic causes of congenital abnormalities,outlined in Table 2, include chromosomal, mono-genic, or polygenic/multifactorial disorders withthe latter being the most frequent genetic cause.Other causes are classified under environmental

Fig. 1 Potter’s sequence

4 A. J. Green and J. J. O’Byrne

agents and include teratogens, maternal illness(e.g., diabetes), and infections. It is important tonote that about 50% do not have any clear causeand most are isolated or non-syndromal. Nonethe-less, parents and families often want an explana-tion as to the origin of their child’s anomaly, and it

is therefore worthwhile to pursue a diagnosiswherever possible. Also accurate etiologicaldetermination can have specific implications fortreatment, prognosis, and assessment of recur-rence risk and counseling of families.

Chromosome Disorders

Disorders of either chromosome number or struc-ture affect about 6/10,000 births and about 6% ofall congenital anomalies are caused by a chromo-some disorder (see Table 2). A finite number ofdisorders of chromosome number exist, and thosemost commonly observed are Down’s syndrome,Edward’s syndrome, and Patau’s syndrome,which will be discussed below. There are poten-tially thousands of disorders of chromosome

Fig. 2 A diagnostic approach to congential anomalies

Table 2 Causes of congenital anomalies

Causes of congenital anomalies Relative frequency

Genetic

Chromosomal 6%

Single gene 7.5%

Multifactorial/polygenic 20–30%

Environmental

Drugs, infections, maternalillness

5–10%

Unknown 50%

Total 100%

Pediatric Clinical Genetics 5

structure, many of which are extremely rare orunique to a particular family. Usually chromo-some disorders are new genetic events in theaffected child; however, a small subset of chro-mosome disorders can be inherited. In these cases,a balanced chromosome rearrangement called atranslocation may present in one or other healthyparent. Chromosome disorders are detected bycytogenetic analysis, and the three most com-monly used analyses are chromosomalG-banding analysis which is often referred to as“karyotyping,” fluorescent in situ hybridization(FISH), and high-resolution array comparativegenomic hybridization (aCGH) more commonlyreferred to as “microarray” (for more details onthese techniques, see section “Genetic Testing”).

Down’s SyndromeDown’s syndrome is the commonest chromosomedisorder, with an average incidence estimated at1 in 700 births and is characterized by the pres-ence of an extra (third) chromosome 21. The mostfrequent congenital anomaly in children withDown’s syndrome is congenital heart disease,usually an atrioventricular defect. However, chil-dren with Down’s syndrome are also more proneto duodenal and jejunal atresias andHirschsprung’s disease. Children with Down’ssyndrome will also have varying degrees of learn-ing difficulty, and it is associated with deafnessand hypothyroidism. There is predominance ofleukemia among patients with Down’s syndromeincluding both acute myeloid leukemia and acutelymphoblastic leukemia. Children with Down’ssyndrome are at a 20-fold increased risk of acutelymphoblastic leukemia. Over 90% of Down’ssyndrome arises as a new genetic event in thechild, with a separate extra chromosome 21.Under these circumstances, it is not necessary tocheck parental chromosomes. Clinicians shouldbe aware of a rare translocation form of Down’ssyndrome which can be readily distinguished onthe child’s chromosomal G-banding analysis, andthe unaffected parents of affected children shouldbe offered chromosomal G-banding analysis asthey may carry a balanced form of a chromosometranslocation.

Patau’s SyndromePatau’s syndrome is caused by the presence of anextra chromosome 13 and is a condition that isusually lethal in the newborn period. It is a rarecondition and occurs in about 1 in 5,000 births.Affected children have congenital heart disease,polydactyly, cleft lip and palate, microcephaly,and often a single frontal lobe in their brain(holoprosencephaly). Like Down’s syndrome,95% are new genetic events. There are rare trans-location forms which can run in families, andagain the distinction between the common andrare forms can be identified on the baby’s chro-mosomal G-banding analysis.

Edward’s SyndromeEdward’s syndrome is caused by the presence ofan extra chromosome 18 and is a condition that isusually lethal in the newborn period. It occurs inabout 1 in 3,000 births. Affected children areusually extremely small and have congenitalheart disease, exomphalos, renal anomalies,clenched hands, and rocker bottom feet. Over95% are new genetic events in the affected infant.

Other Chromosome DisordersKlinefelter’s syndrome is a condition character-ized by the presence of an extra X chromosomein a male, i.e., 47,XXY. Boys with Klinefelter’ssyndrome rarely present surgically in childhood.However, a significant number can have delayedpuberty, teenage gynecomastia, or fertility issuesin later life.

Turner’s syndrome is the presence of a single Xchromosome in a female, i.e., 45,X. Girls with Tur-ner’s syndrome are more prone to congenital heartdisease, classically coarctation of the aorta, and alsohave renal anomalies. They may present in the new-born period with these anomalies in the presence orabsence of “puffy hands and feet” caused by lymph-edema. Short stature is a feature and spontaneouspuberty is unlikely, due to ovarian dysgenesis.

Di George or velocardiofacial syndrome is usu-ally caused by a very small deletion of one ofchromosomes 22 which is detectable by FISH.A wide range of congenital anomalies may bepresent, most commonly congenital heart disease.

6 A. J. Green and J. J. O’Byrne

Cleft palate and laryngeal and renal anomalies mayalso be present in association with hypocalcemia,learning difficulties, and immunodeficiency.

Single-Gene Disorders

Single-gene or monogenic disorders are caused byan alteration in one or both copies of one specificgene. Over 4,000 single-gene disorders have beendescribed, and about 7.5% of all congenital anom-alies are caused by a single-gene disorder (seeTable 2). Single-gene disorders can be dividedinto groups via their inheritance pattern of whichthere are three principal modes: autosomal domi-nant, autosomal recessive, and X-linked reces-sive. Other rare patterns of inheritance includeX-linked dominant, mitochondrial inheritance,triplet repeat disorders, and genetic imprinting.The various single-gene disorders are discussedunder these headings below.

Autosomal Dominant InheritanceAutosomal dominant disorders are caused by amutation in one of the two copies of a specificgene. Families will often have several generations

affected, and usually the number of males andfemales affected is approximately equal. The hall-mark of autosomal dominant inheritance is fatherto son transmission. Each child born to a personwith an autosomal dominant disorder has a 50:50chance of inheriting the gene mutation responsi-ble for the condition and is thereby predisposed todeveloping the condition (see. Fig. 3). However,not all people with an autosomal dominant disor-der have a family history of the condition; somepeople can represent a newmutation in the specificgene for their condition. For some autosomal dom-inant conditions such as Marfan’s syndrome orneurofibromatosis type 1 (NF1), the new mutationrate can be 20–30%. Autosomal dominant disor-ders also often display variability in both pene-trance (whether a person develops any sign of thecondition) and expression (how the conditionman-ifests). For example, NF1 will almost always man-ifest in someone who has an altered NF1 gene, butdifferent people with NF1 can manifest the condi-tion in different ways, with some people showingmild expression with a few skin lesions and otherswith severe intracerebral complications. Thismeans that NF1 is almost completely penetrantbut the expression of the condition is very variable.

Fig. 3 Autosomaldominant inheritance

Pediatric Clinical Genetics 7

In contrast, only 80% of people who have a singlealtered gene for the rare hereditary form of retino-blastoma will actually develop an eye tumor. Thepenetrance in this situation is 80%, but the expres-sion of the altered gene is consistent, as manifestedby a retinoblastoma.

Autosomal dominant disorders are not com-monly seen in neonatal surgical practice; how-ever, examples that may be observed in familieswith reduced penetrance include Hirschsprung’sdisease, Beckwith-Wiedemann syndrome, andpyloric stenosis. Some forms of craniosynostosisand orofacial clefting can also be a result of anautosomal dominant disorder. Rare childhoodcancer predisposition syndromes such as poly-posis coli, retinoblastoma, and Li-Fraumeni syn-drome are inherited in an autosomal dominantmanner.

Single-Gene Disorders and AutosomalRecessive InheritanceAutosomal recessive disorders are caused whenboth copies of a particular gene responsible for thecondition are altered. Both parents would carrythe condition and be unaffected as each parentwould have one normal and one altered gene. Inmost cases, carrying an autosomal recessive con-dition has no effect on the person. Two of thechild’s four grandparents are likely to carry thecondition, and it is probable that many of thepatient’s relatives would unknowingly carry thecondition.

When both parents carry an alteration in thesame autosomal recessive gene, there is a 25% orone in four chance to each of their children ofinheriting both altered copies and probably thecondition (see Fig. 4a). A child born to a personaffected by an autosomal recessive disorder willautomatically carry the condition (see Fig. 4b).The risk to a child, born to a couple, one ofwhom is a confirmed carrier of the condition andone of whom is not, depends on the populationrisk that the second parent is a carrier for analteration in the same gene.

Autosomal recessive disorders are commonlyobserved in pediatric practice, and some mayrequire surgery in the neonatal period. The fre-quency of the autosomal recessive disorders

encountered depends on the patient population.Each regional population has its own recessivedisorders, where the frequency of carriers forthose disorders is highest. For instance, cysticfibrosis (CF) is a very common autosomal reces-sive disorder in Western Europe, whereas sicklecell anemia is the commonest autosomal recessivedisorder in West Africa. Some other examples ofautosomal recessive conditions are beta-thalasse-mia, spinal muscular atrophy, several of themucopolysaccharidoses, and congenital adrenalhyperplasia. Direct genetic diagnosis is availablefor many of these conditions.

Many countries have a newborn screening pro-gram which includes testing for a number of auto-somal recessive disorders including galactosemia,homocystinuria, sickle cell disease, and CF. Also,among some populations, carrier testing for cer-tain autosomal recessive diseases is available tocouples planning a pregnancy or to women in theearly stages of pregnancy.

Single-Gene Disorders and X-LinkedRecessive InheritanceX-linked conditions are caused by alterations in agene on the X chromosome. Classic examples ofsuch conditions include hemophilia A and B,Duchenne and Becker muscular dystrophy, andHunter’s syndrome. Males have only one X chro-mosome, and therefore those with an altered genemanifest the disease, as there is no normal copy ofthe gene to compensate. Females have two Xchromosomes, and therefore, even if a femalehas an altered gene, the normal gene usually off-sets the effect of the altered gene. Females cantherefore carry an X-linked condition, while onlysome females will manifest the disorder. Thedaughters of a man with an X-linked recessivecondition are all obligate carriers, as they allinherit his X chromosome. The sons of a manwith an X-linked condition are all normal, asthey inherit his Y chromosome and not his Xchromosome (see Fig. 5a). When a woman is acarrier of an X-linked condition, each of her sonshas a 50:50 chance of being affected, and each ofher daughters has a 50:50 chance of being a carrier(see Fig. 5b). There can be a relatively high muta-tion rate for some X-linked recessive conditions,

8 A. J. Green and J. J. O’Byrne

and affected boys may not have any family historyof the condition. Approximately one-third of boyswith the X-linked condition Duchenne musculardystrophy occur as a result of new mutations.

Single-Gene Disorders and AtypicalForms of InheritanceX-linked dominant inheritance is a much rarerinheritance pattern of single-gene disorders thanthose discussed above. It can be difficult to

Fig. 4 Autosomalrecessive inheritance

Pediatric Clinical Genetics 9

distinguish from autosomal dominant inheritancewhen observing of the family tree with the excep-tions that females will be more mildly affected andmale-to-male transmission does not occur. An

example of an X-linked dominant condition ishypophosphatemic rickets.

Mitochondrial inheritance is a very unusualpattern of inheritance and observed in inheriteddiseases caused by single-gene disorders of the

Fig. 5 X linked recessiveinheritance

10 A. J. Green and J. J. O’Byrne

mitochondrial genome. Most of the proteins nec-essary for the mitochondrial function and struc-ture are encoded for by the nuclear genome and sofollow the standard Mendelian inheritance pat-terns. Mitochondria however also contain theirown small genome of 18 kilobases, with manycopies per cell as each cell contains many mito-chondria. The mitochondrial genome does notfollow the Mendelian patterns of inheritance andreplicates independently and far more frequentlythan the nuclear genome. Several important mito-chondrial proteins are encoded by the mitochon-drial genome, but mitochondria are only inheritedvia oocytes and not sperm. Therefore, where agene alteration is in the mitochondrial genome, itwill pass exclusively down the female line, butboth males and females can be affected. The chil-dren of an affected male will not inherit his mito-chondrial gene alteration. Children withmitochondrial disorders usually display multi-systemic involvement and can present with variedsymptoms at any age, including myoclonic sei-zures, acute acidosis, muscle weakness, deafness,or diabetes. A number of point mutations anddeletions in the mitochondrial genome have beendescribed in patients with a wide variety of con-ditions, including MELAS (mitochondrialencephalopathy with lactic acidosis and stroke-like episodes) or MERRF (myoclonic epilepsywith ragged red fibers on muscle biopsy). Tocomplicate matters further, Leber’s hereditaryophthalmopathy is a mitochondrially inheritedcondition, with a characteristic mutation in themitochondrial genome, but the expression appearsto have an X-linked recessive influence.

Triplet repeat expansions can cause single-gene disorders in conditions such as fragile Xsyndrome, Huntington’s disease, Friedreich’sataxia, and several forms of spinocerebellarataxia. With these conditions, a gene is considerednormal when it has a small stable number of threeDNA (triplet) repeat copies (e.g., 20 C-A-Grepeats). The person is unaffected as the genefunctions normally and the children of that personinherit the same number of repeats in their geneand so are unaffected too. If the gene has a largernumber of repeats than normal, then the function

of the gene and encoded protein may becompromised and person may become affected.This instability of the repetitive element mayresult in even further expansion in the next gener-ation and the affected children of that person mayhave more serious disease. This phenomenon,where a condition appears to worsen from gener-ation to generation, is known as anticipation. Oneof the most extreme examples of this molecularmechanism is observed with congenital myotonicdystrophy, where a minimally affected mother canhave a profoundly affected infant as a small unsta-ble repeat expansion in the mother increases tomany hundreds of repeats in her affected infant.

Genetic Imprinting is demonstrated by condi-tions such as Prader-Willi syndrome, Angelman’ssyndrome, Russell-Silver syndrome, Beckwith-Wiedemann syndrome, and the rare condition oftransient neonatal diabetes mellitus. Surgicalinput may be required in the neonatal period forsome of these conditions (e.g., exomphalos repairin Beckwith-Wiedemann syndrome).

In genetic imprinting, an imprinted genebecomes marked during meiosis to indicate theparent from which it is inherited. For some genes,it appears to be important not only to inherit twocopies of that gene but to inherit one from eachparent. Some genes may be silenced, dependingupon which parent has passed on that gene. Anexample of imprinting is demonstrated when asmall deletion of chromosome 15q occurs.Depending upon whether the deletion occurs onthe maternally or paternally derived chromosome15, a different effect is observed. If the deletionoccurs on the chromosome inherited from achild’s normal father, the child will developPrader-Willi syndrome (PWS). If the same dele-tion occurs on the chromosome inherited from achild’s normal mother, the child will develop aclinically distinct condition, Angelman’s syn-drome (AS). In addition, if a child inherits twocopies of chromosome 15 from the one parent,uniparental disomy (UPD), the child will developPrader-Willi or Angelman’s syndrome (maternalUPD results in PWS, while paternal UPD resultsin AS). The genes in this area of chromosome15 are therefore said to be “imprinted.”

Pediatric Clinical Genetics 11

Polygenic/Multifactorial Disorders

Polygenic/multifactorial gene disorders are disor-ders that do not have a clear mode of inheritance,where a disease may arise as a result of the effectsof several genes and/or be influenced by severalenvironmental factors. It is estimated that thesepolygenic disorder conditions cause up to 30% ofcongenital anomalies (see Table 2). Examples of acongenital anomaly that is thought to follow poly-genic inheritance are neural tube defects such asspina bifida or anencephaly. A threshold effect isthought to exist where hypothesized genetic fac-tors, known environmental factors (e.g., inade-quate maternal folic acid levels in the firsttrimester), sand other chance factors combine toresult in a neural tube defect. Another example iscleft lip and palate, which usually occurs in theabsence of a family history. However, monozy-gotic (identical) twins have a high concordancefor cleft palate, and the recurrence risk in anotherchild is known to be higher than the populationrisk suggesting a genetic influence. However, thegenetic influence does not follow a typical Men-delian or single-gene pattern of inheritancesuggesting that other factors are often necessaryfor the condition to manifest. Other examples ofpolygenic disorders include congenital heart dis-ease, non-syndromic Hirschsprung disease,vesico-ureteric reflux, and coeliac disease.

Genetic Testing

Chromosomal G-Banding Analysis(Standard Karyotyping)

Chromosomal G-banding analysis is now oftenreferred to a “karyotyping.”

Indications for carrying out a standard chromo-somal G-banding analysis in a neonate with con-genital anomalies are if signs suggestive of aknown chromosome disorder such as Down’s orPatau’s syndrome are observed, or if the baby has asuspected disorder of sexual development, wherethe chromosomal gender is important to identify.Previously, this would have been considered the

first-line test of a child with multiple mal-formations, but now the recommended first-linetest in an infant or older child with multiplemalformations is high-resolution array CGH (seesection “Array Comparative Genomic Hybridiza-tion/Chromosomal Microarray Analysis”).

To examine chromosomes using chromosomalG-banding analysis, dividing cells (usually lym-phocytes, amniotic fluid cells, or fibroblasts) inculture must be examined. Cells are arrested inthe metaphase stage of mitosis where the chromo-somes are condensed and easily visualized. Using“Giemsa” stain, a characteristic positive and nega-tive “G”-banding pattern is observed on each chro-mosome. Each chromosome has a constriction,called a centromere, dividing the chromosomeinto a short (p) arm and a long (q) arm. Each armhas a number of prominent bands, which can thenbe subdivided into smaller bands. For example, thegene for the ABO blood group is localized tochromosome 9q34. The gene thus lies in the fourth

sub-band from the centromere (q34) of the third

band from the centromere (q34) on the long arm

(q34) of chromosome 9 (9q34).Chromosome abnormalities can broadly be

classified into abnormalities of chromosome num-ber or structure. The critical issue in most cases fordetermining the significance of a chromosomeabnormality is whether the abnormality givesrise to an excess or deficiency of the normal dip-loid state (aneuploidy).

Abnormalities of chromosome number are rel-atively common, but many are not recognized, asthey may result in the early loss of a pregnancy.Triploidy (69 chromosomes) and tetraploidy(92 chromosomes) are relatively common causesof early pregnancy loss. Trisomy, the presence of asingle extra chromosome (47 chromosomes), isalso a common cause of miscarriage. Specifictrisomies can give rise to children with manycongenital anomalies, the commonest being tri-somy 21 (Down’s syndrome), trisomy 13 (Patau’ssyndrome), and trisomy 18 (Edwards’ syndrome)which are discussed in more detail in sections“Down’s Syndrome,” “Patau’s Syndrome,” and“Edward’s Syndrome.” All these trisomies usu-ally occur as a result of autosomal non-disjunction

12 A. J. Green and J. J. O’Byrne

in meiotic division of the oocyte. Innon-disjunction, the specific chromatids fail toseparate, resulting in an extra chromosome inone oocyte, and it tends to occur more frequentlywith increasing maternal age.

Abnormalities of chromosome structure canoccur in many ways including inversions, inser-tions, duplications, deletions, isochromosomes,ring chromosomes, and translocations. Inversions,both pericentric and paracentric, are usually bal-anced and inherited without any phenotypic effect.For a person who carries a balanced paracentricinversions, there is usually a low risk of producinga live-born baby with an unbalanced chromosomerearrangement, but pericentric inversions maycarry a higher risk. Insertions, duplications, dele-tions, isochromosomes, and ring chromosomes areall usually aneuploid and often associated withsignificant clinical abnormalities.

Two types of translocations occur, reciprocal andRobertsonian. Reciprocal translocations occurwhere there is exchange of genetic material fromone arm of a chromosome in return for geneticmaterial from a different chromosome. These areusually balanced, without any clinical effect, butmay again carry a risk of having a child with con-genital anomalies due to an unbalanced karyotype.Robertsonian translocations occurs between theacrocentric chromosomes (i.e., chromosomes13–15, 21, and 22), where there is no appreciablecodingmaterial on a very small short (p) arm. Thesetranslocations can run in families, and those whocarry a Robertsonian translocation involving chro-mosomes 21 or 13 may be at significantly higherrisk of having a child with Down’s or Patau’s syn-drome as an unbalanced product of the transloca-tion. The distinction between the rare translocationform of these conditions and the more commonnon-disjunction form can be identified on the child’schromosome G-banding analysis.

Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) uses spe-cific fluorescently labelled small DNA fragmentsor probes corresponding to 40–50 kb of DNA fromspecific regions of chromosomes which allows for

targeted higher resolution analysis of specific chro-mosomes. An example where this technologywould be commonly used is testing for the submi-croscopic deletion of chromosome 22q11 whichoccurs in most cases of Di George syndrome.

FISH can also be carried out to get a rapidanalysis for numerical chromosome anomaliessuch as Down’s syndrome, without the need forculturing blood cells, by analyzing interphasenuclei with a FISH probe containing chromosome21 material. FISH testing can also be used to givea rapid determination of the chromosomal genderusing X and Y probes in interphase nuclei.

Array Comparative GenomicHybridization/ChromosomalMicroarray Analysis

Array comparative genomic hybridization (arrayCGH) is a relatively new technology employed inclinical practice and a form of molecularkaryotyping that can detect losses (deletions) orgains (duplications) of chromosomal segments orvariations in the expected number of copies ofDNA in certain segments of the genome known ascopy number variations (CNVs) at a resolution of atleast 100 times greater than conventional G-bandingchromosome analysis. This technology is oftenreferred to a “microarray,” but it is important to beaware that many different types of “microarray”exist such as single-nucleotide polymorphism(SNP) array as well as mRNA, protein, and tissuemicroarrays.ArrayCGHandSNParray are themostcommonly used array types in the clinical setting,while the others are used mainly in the researchworld to look for variants of individual genes,protein-protein interactions, and the molecular biol-ogy or immunohistochemistry of the samples.

Array CGH compares the patient’s DNA tocontrol DNA using two different fluorescentlabels (see Fig. 6). Labelled control (red) andpatient (green) DNA arrangements are hybridizedto an array (chip) containing oligonucleotide pro-bes (DNA sequences from genes throughout thehuman genome). A digital imaging system is usedto capture and quantify the fluorescence ratiowhich indicates any differences in the patient’s

Pediatric Clinical Genetics 13

genome compared to the control genome. Whenonly the control DNA is present in a region, theabsence of patient DNA (deletion) is indicated bya red fluorescent dot. When there is more patientthan control DNA (duplication), the patient labelis over replicated and is indicated by a green dot.When there are no copy number changes, thereshould be equal amounts of control-labelled andpatient-labelled DNA (yellow dots).

Array CGH analysis should be considered ininfants born with multiple malformations or signif-icant dysmorphology. Genomic array technologywill identify pathogenic chromosomal anomaliesin up to 30%of infantswithmultiplemalformations,and it is reported the likelihood of an abnormal arrayCGH increases with the number of clinical abnor-malities, i.e., patients with �4 clinical variableshave demonstrated a 30% incidence of abnormalchromosomal microarray findings compared with~9% of patients with �3 clinical variables.

Array CGH has advantages over chromosomalG-banding analysis and FISH. Array CGH maydetect DNA imbalances much more precisely than

chromosomal G-banding analysis and can revealwhich specific genes are included in the deletionor gain. Array CGH can detect chromosomeimbalances when there are no clues to what thechromosome anomaly might be and so would notbe detected by performing specific FISH. It canfurther define breakpoints and the size of theimbalances in certain cases.

Although array CGH is more detailed thanstandard chromosomal G-banding analysis, ithas not replaced G-banding in all situations.Array CGH will not identify balanced chromo-some rearrangements such as balanced transloca-tions and inversions as these do not result in anyloss or gain of chromosome material. Array CGHwill also not detect some types of polyploidy suchas triploidy or low-level mosaicism (<20%). Achromosomal G-banding analysis would be nec-essary to detect these balanced anomalies andaneuploidies and in situations where mosaicismis suspected would be the preferred primary test.Array CGH will also not detect point mutations orepigenetic abnormalities.

Red = DeletionGreen = DuplicationYellow= No change/Normal

Control DNAPatient DNA

Array containing oligonucleotides

This should really be single stranded and fragmented…

Fig. 6 Array CGH analysis

14 A. J. Green and J. J. O’Byrne

The results of microarray testing are oftencomplex and require confirmation and carefulinterpretation, often with the assistance of a clin-ical geneticist.

Three possible results may be attained from arrayCGH: (a) a normal result with no clinically signifi-cant variation, (b) a definitely abnormal result with aknown pathological variation, and (c) a variation ofunknown significance (VUS).VUSare oftenCNVs.Every person has approximately 100 CNVs whichcan affect numerous genes but are only sometimespathogenic. It may be difficult to establish the sig-nificance of someCNVs, and targeted parental arrayCGHs are usually recommended in the first instanceto help with the interpretation.

Specific Testing for Common KnownSingle-Gene Mutations

Laboratory tests for single-gene disorders havebeen available for a considerable amount oftime. Tests such as hemoglobin electrophoresisfor sickle cell anemia and thalassemia or enzymeassays for Tay-Sachs’ disease are examples oftests that predate the genetic testing era andremain very effective in resolving clinical issuesin individual families. Increasingly specificDNA-based tests are being used in diagnosis andprediction of single-gene disorders.

The two major techniques used in specificmolecular genetic analysis are the polymerasechain reaction (PCR) and the less frequentlyused southern blotting.

PCR is a technique which allows amplificationof a specific genetic region in large quantities froma small amount of DNA template. Using oligonu-cleotide primers, DNA generated by PCR can beused to detect known pathogenic DNA mutationsand give a diagnosis, even without any knowledgeof the patient’s clinical status, e.g., the PCR detec-tion of the Phe508del deletion in both copies of aperson’s cystic fibrosis transmembrane regulator(CFTR) gene immediately gives a diagnosis ofCF. Similarly a PCR test detects a deletion ofexons seven and eight in both alleles of a genecalled SMN on chromosome 5q in almost allchildren with spinal muscular atrophy. If the

search is for an unknown DNA mutation, suchas those seen in hereditary breast and ovariancancer, then many pieces of DNA generated byPCR can be sequenced and analyzed using anautomated DNA analyzer.

Southern blot analysis of DNA from infantswith congenital myotonic dystrophy shows a verylarge expansion in a triplet repeat DNA sequencein the myotonin kinase gene on chromosome 19.

Next-Generation Sequencing and GenePanels

With the advent of massive parallel sequencing, ornext-generation sequencing, it is now possible tosequence large sections of genomes in a short timeframe. These rapid technological advances alongwith the continued identification of pathogeniccausing genes mutations and the improvement ingenotype-phenotype correlations have led to thedevelopment of “gene panels” for hundreds of con-ditions. Gene panels are now available to investigatechildren with specific malformations. Examples ofconditions that gene panel testing is now availablefor include arthrogryposis, anophthalmia, cranio-synostosis, non-syndromic Hirschsprung’s disease,holoprosencephaly, lissencephaly, overgrowth,skeletal dysplasias, vascular malformations, andpolycystic kidney disease.

Although these panels are currently expensive,prices are expected to fall, and in the long run,targeted gene panel testing, led by an experiencedclinical geneticist, can be a low-cost, time-effectivetest particularly if the suspected disorder has aphenotype that could be caused by a number ofgenes. One such example of a very useful genepanel is one that is applicable to disorders of theNoonan syndrome spectrum which contain condi-tions such as Noonans, lentigines, electrocardio-graphic conduction defects, ocular hypertelorism,pulmonary stenosis, abnormalities of the genitals,retarded growth and deafness (LEOPARD),cardiofaciocutaneous, and Costello syndromes.These are a genetically heterogeneous group ofautosomal dominant disorders that often haveover lapping clinical features and can be difficultto differentiate. The gene panel now contains up to

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11 genes (BRAF, KRAS, HRAS, NRAS,MAP2K1, MAP2K2, PTPN11, RAF1, CBL,SOS1, SHOC2) which can help distinguish whichcondition is present. Another example of a genepanel with a high clinical utility is available is thatfor Meckel Gruber syndrome which is the mostcommon syndromic form of neural tube defect. Itpresents with a classic triad of clinical features ofoccipital encephalocele, cystic kidneys, andfibrotic change of the liver, but the phenotypemay also include features such as postaxial poly-dactyly, skeletal dysplasia, microphthalmia, genitalanomalies, cleft lip and palate, and heart defects,and any one of a number of genes (CC2D2A,CEP290, MKS1, RPGRIP1L, TCTN2,TMEM216, TMEM67) may be causative.

Gene panel tests are constructed, analyzed, andreported with an intentional blindness to all, but aspecifically selected list of genes and cliniciansshould be cognizant of which genes were notreported when interpreting a test result. Panel testare also more likely to identify a VUS than adeleterious mutation. In addition not all genesincluded in panel tests are unequivocally linkedto the disease/phenotype, and for most genes, thepenetrance is highly variable making it challengingto translate a specific mutation into an absolutecondition risk.

In time to come, we would expect that panelgene testing will become standard for children ofall ages presenting with malformations.

Conclusions and Future Directions

As the capacity for diagnosis of genetic conditionscontinues to develop at a rapid pace, medical sub-specialties, traditionally rarely aligned with clini-cal genetics, are encountering on a far morefrequent basis patients with genetic diagnoses.This requires the clinicians working in specialitieslike pediatric surgery to keep abreast of develop-ments in the area of clinical genetics, a challengenot easily surmountable in this era of such rapidtechnological advance. Array CGH was added tomainstream clinical practice in the last decade,while gene panels are currently available for hun-dreds of different conditions and the list in

expanding on an almost daily basis. Soon theability to examine the coding regions of all23,000 genes with exomic sequencing will bestandard in clinic practice and will eventually befollowed by whole genomic sequencing.Although this exciting new technology willincrease the diagnostic rate of genetic disorders,it will bring a new set of challenges such as anincreased number of VUS and the ethical dilemmaof reporting variations in genes not associatedwith the phenotype under investigation.

Noninvasive prenatal screening is another newgenetic technology that will influence neonatalsurgical practice. It is now widely available forthe investigation of trisomies 21, 18, and 13 and isbeing expanded to screen for many other geneticconditions. This will increase the number of pre-natal diagnoses which will allow for early prepa-ration of required surgical management in thenewborn period or even in utero.

Pediatric and in particular neonatal surgicalpractice will continue to become more involvedwith the subspecialty of clinical genetics as thesurgeons encounter on a more frequent basis casesand conditions with a known genetic. The tech-nology to investigate the genetics of these condi-tions is becoming available to an increasing breathof specialties including pediatric surgery. Closeliaison between surgeons and clinical geneticistswill be essential in managing these investigationsand diagnoses correctly and overcoming theassociated challenges.

Cross-References

▶Antenatal Diagnosis▶Anorectal Malformations▶Choanal Atresia▶Congenital Airway Malformations▶Congenital Biliary Dilatation▶Congenital Diaphragmatic Hernia▶Congenital Esophageal Stenosis▶Congenital Malformations of the Lung▶Duodenal Obstruction▶Embryology of Congenital Malformations▶Esophageal Atresia▶Gastroschisis

16 A. J. Green and J. J. O’Byrne

▶Omphalocele▶Macroglossia▶ Pierre Robin Sequence▶ Prune Belly Syndrome▶Urethral Anomalies▶Urogenital Sinus and Cloacal Anomalies▶The Bladder Exstrophy, Epispadias, CloacalExstrophy

▶Tissue Engineering and Stem Cell Research

Further Reading

OMIM, Online Mendelian Inheritance in Man – a databaseof human genes and genetic disorders developed bystaff at Johns Hopkins www.ncbi.nlm.nih.gov/Omim/

Orphanet – a database (in several languages) of geneticdisorders, clinical information, clinic listings andresearch and diagnostic genetic testing for a widerange of disorders www.orpha.net

Contact a Family – a UK charity for families with disabledchildren, which offers information on specific condi-tions and rare disorders. www.cafamily.org.uk

Understanding Gene testing (n.d.) – a website with infor-mation on basic genetic concepts, and the utility andlimitations of genetic testing http://www.accessexcellence.org/AE/AEPC/NIH/

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