GeNeViSTA Genetic Approach to Congenital Malformationsiamg.in/genetic_clinics/adm/articlepdf/July-September-2018-04-gene... · Table 3 Points to be noted in the history in the clinical

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Genetic Approach to Congenital MalformationsShubha R Phadke

Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

Correspondence to: Dr Shubha R Phadke Email: [email protected]

Introduction

Development of a multicellular organism from asingle cell is a very complex, co-ordinated pro-cess governed by many genes and their timelyexpression in various organs at various stages ofembryogenesis. It is not surprising that in someembryos some things go wrong during develop-ment leading to structural abnormalities of variousorgans. Major defects in very early stages ofembryogenesis may not be compatible with sur-vival and lead to spontaneous abortions. Most ofthe structural abnormalities/anomalies also knownas malformations of internal or external organsoccur during the first trimester (dysmorphogene-sis), though they may be detected in the secondtrimester of pregnancy by fetal ultrasonographyor after birth during the neonatal period or ininfancy. Some malformations involving abnormal-ities of the size of an organ like microcephalyor obstruction causing hydronephrosis or hydro-cephalus may become obvious during the laterpart of pregnancy or during infancy. Though allthe structural defects are congenital, i. e. presentsince birth, internal anomalies may not be detectedtill they manifest with some symptoms. Malfor-mations of internal organs can be diagnosed byvarious imaging techniques like ultrasonography,echocardiography, CT scan or Magnetic ResonanceImaging (MRI) which are used when there are somesymptoms indicating the possibility of internal mal-formations. Individually each malformation is rarebut 3% of neonates have one major malformationand around 0.7% of neonates have multiple mal-formations. This is a brief review about the variousbirth defects and malformations and a systematicclinical approach to diagnose the same.

Types of Birth Defects

The various types of structural abnormalities oforgans are given in Table 1 and Figure 1.

Figure 1 [a] Meningo-myelocele – a major malfor-mation [b] Hypertelorism and upslantof eyes in a child with characteristicfacial phenotype of a child with Downsyndrome due to trisomy 21[c] Ecto-dermal dysplasia showing absence ofteeth and sparse, light coloured hair[d] Polydactyly – a minor malformation[e] Talipoequinovarus – an example ofdeformation.

Patterns of Congenital MalformationsA malformation or structural defect of an organcan be an isolated anomaly or may be associatedwith major or minor anomalies or variants of othersystems. Multiple malformations are classified intosyndromes, sequence and association. This givesan idea about the etiopathogenesis and is useful inclinical approach to the management.• Syndrome: A syndrome is often used to

describe a pattern of malformations occur-ring together and definitely or presumablycaused by a common etiology. The syndromemay be due to a chromosomal etiology (e.g. Down syndrome due to trisomy 21), amicrodeletion syndrome (e. g. Williamssyndrome due to a submicroscopic deletion

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Table 1 Types of Birth Defects.

Type of birth defect Description Examples CommentMajor malformation(single major anomalyin 3% of births,multiplemalformations in 0.7%neonates)

Morphological defectsthat occur due toerrors in the normaldevelopment anddifferentiation of anembryo.

Meningocele, posteriorurethral valve,ventricular septaldefect, cleft lip

If untreated, will lead tosignificant impairment ofthe function of the organleading to mortality ormorbidity

Minor malformation(14% and 3% ofneonates have a singleand multiple minormalformationsrespectively)

Polydactyly, ear tag,Single palmar crease

Only of cosmeticimportance, but of use indiagnosis of etiology

Normal variants Clinical features whichare at the far end ofnormal distribution

Low set ears,hypertelorism

May be present in normalindividuals but inassociation with other birthdefects can be of help inthe diagnosis of asyndrome

Deformation Abnormal shape of anormally formedstructure due todistortion caused bymechanical forces

Talipes equinovarus ina child with amyoplasiaor congenitalmyopathy due tooligohydramnios orintrauterine crowding

May have good prognosiswith postnatal intervention.Risk of recurrence in thefamily is usually notincreased if the causativefactor does not recur

Disruption Damage or dissolutionof a part followingnormal development

Amputation of digitsdue to damage byconstricting amnioticband orthrombo-embolicphenomenon

Usually do not recur in thefamily

Dysplasia Morphological defectscaused by abnormalmaturation andorganization of cellsinto tissue

Achondroplasia(involvement ofbones), Ectodermaldysplasia (hair, teeth,sweat glands and nailsare involved)

Involvement of multipleorgans in the body wherethe particular tissue ispresent. Mostly are singlegene disorders.

on chromosome 7q11), or mutation in a gene(eg. Apert syndrome due to mutation in FGFR2gene).• Sequence: A single malformation may lead toa chain of events leading to multiple anoma-lies and such a group of anomalies is knownas sequence. An example is congenital ab-sence of kidneys leading to oligohydramnios;which in turn leads to flattening of face, lunghypoplasia and clubfeet, called the Pottersequence.

• Association: The co-occurrence of a groupof malformations together more frequentlythan can occur by chance, without an iden-tified common etiology, is labelled as anassociation. As genomic techniques areidentifying etiologies, some of the cases pre-viously described as associations are beingregrouped as syndromes. E.g. some casesof CHARGE (Coloboma, Heart defect, Atresiachoanae, Retarded growth, Genital and Earanomalies) association are now found to bedue to mutation in the CHD7 gene.


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Clinical Presentation of MalformationsMany malformations such as neural tube defectsmanifest at birth either because they are obvi-ous on external examination or may present withsymptoms due to the resulting functional defecte.g. tracheoesophageal fistula. Some internalanomalies like atrial septal defect, hydronephrosisdue to pelviureteric junction obstruction or ec-topic kidney may go undetected for many years.Nowadays, many major malformations are get-ting detected prenatally due to routine antenatalultrasonographic evaluation. Some fetuses withmalformation(s) may get spontaneously aborted orare stillborn. A neonate with a major malformationis an emergency and evaluation for associatedmajor or minor malformations for making anetiological diagnosis is essential in addition tosupportive care and appropriate curative surgery.

Importance of Etiological Diagnosis ofCongenital Birth DefectsMost of the congenital birth defects are genetic inorigin; though in some cases the definite geneticdefect is not still identified. Other causes areteratogens. For some malformations, especiallyisolated malformations like neural tube defectsand pyloric stenosis, multifactorial etiologies en-compassing multiple genetic variations interactingwith environmental factors like folic acid intake arehypothesized. One type of congenital malforma-tion may be caused by different etiologies and theetiological diagnosis can be made by evaluatingfor associated features and genetic tests. Table2 shows one example where the enlargement oflateral ventricles in brain can be due to variouscauses and risk of recurrence depends on theetiology. Figure 2 shows brain imaging showing

Table 2 Etiologies associated with ventriculomegaly.

Disorder Etiology Risk of recurrence insibling

Comment

Aqueductalstenosis

Monogenic - CCDC88Cgene (Autosomalrecessive)

25% Clinically may not bedifferentiable from X- linked

Aqueductalstenosis

Monogenic- L1CAM gene(X - linked)

50% of male offspring,if mother is a carrier

Adducted thumb, ifassociated, is confirmatory ofX- linked variety

Retinoic acidintake by mother

Teratogenic Nil, if drug intake isavoided

History of intake by motherduring pregnancy

Cytomegalovirus(CMV) infectionin fetus

Transplacentaltransmission of virusfrom infected mother(History of symptoms ofinfection in mother mayor may not be present)

Nil as recurrence orreactivation of infectionin mother does notcause symptomaticinfection in the fetus

Brain imaging may suggestthe diagnosis due toperiventricular calcification.Molecular testing for CMVvirus is confirmatory

Aase-Smithsyndrome

Autosomal dominant Nil if parents arenormal

Cleft palate, jointcontractures, Dandy Walkermalformation

Lissencephaly Many different causes Depends on etiology MRI brain confirmslissencephaly

Hydrolethalussyndrome

Monogenic - HYLS1 orMPDZ gene (Autosomalrecessive)

25% Polydactyly, cleft lip, cardiacanomalies are seen

Holoprosen-cephaly

Many different causesincluding chromosomal,single gene, syndromicetiologies

Depends on etiology Carrier parent of a singlegene mutation may have asingle central incisor as avery mild feature (formefruste) of holoprosencephaly


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a) b)

c) d)

Figure 2 Neuroimaging showing enlargement of brain ventricles due to different etiologies [a] DandyWalker malformation [b] X linked aqueductal stenosis associated with hypoplastic palmarflexed thumbs [c] Pseudo-TORCH syndrome showing periventricular calcification in a fetusfrom a consanguineous family with a previous child diagnosed with ventriculomegaly [d]Walker-Warburg syndrome with lissencephaly, microphthalmia and cleft lip.

dilatation of ventricles of the brain due to differentetiologies.

As seen in Table 2, risk of recurrence of themalformation and the prognosis varies dependingon the etiology. Etiological diagnosis also helpsin early detection of treatable complications bysurveillance and in assessing the risk of recurrencein subsequent offspring in the family.

Clinical Approach to a Patient with Con-genital Malformation(s)A patient, usually a child or a neonate is broughtfor evaluation due to presence of a major malfor-mation or due to presence of multiple anomalies(often it may be just different looking facial featuresor facial dysmorphism) which may or may not beassociated with developmental delay/ intellectualdisability or other neuro-developmental disorderslike autism. The clinician should evaluate for asso-ciated major malformations, functional disabilitieslike developmental delay or deafness and mostimportantly attempt to identify the etiology. The

process of diagnosis may be long drawn in manycases. The clinician needs to take into account theunderstanding and expectations of the parents,their psychosocial and educational backgroundand talk to the family with empathy, so that thefamily is convinced about the desire of the clinicianto help the patient. Good communication with thepatient and family helps to give them confidenceand get their co-operation and commitment whichare needed for the evaluation, which often involvesa series of tests, some of which may not giveany diagnostic results. Children with dysmorphicfeatures and their parents may be sensitive to the‘different’ looks and affected individuals often havea poor body image. The dysmorphic features areof diagnostic importance and need to be notedby careful examination of the affected child andsometimes parents also. However, during the clin-ical examination, utmost care should be taken notto hurt the family by using unfriendly/ insensitivedescriptive terms. Photographic documentation isimportant but should be done only after writtenconsent of the patient or the parents.


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Clinical EvaluationThe clinical approach to a child with malformationor dysmorphism includes detailed clinical historyand thorough head-to-toe and systemic examina-tion. The prenatal history and family history playvery important roles (Table 3).

Examination of a dysmorphic childThe initial part of evaluation is anthropometricmeasurement of the height / length, weight andhead circumference and comparison with age/sexmatched controls. The head-to-toe examinationshould be carried out by careful evaluation af-ter removing clothes. Facial dysmorphism, hairpattern, nails, neck, fingers and toes, body hair,and pigmentary abnormalities need to be noted.Some features may be present in either of theparents and suggest that the feature is a familialcharacter and may not be related to the etiologyof developmental delay / intellectual disability ora syndrome. The American Journal of MedicalGenetics had published a special issue on thedefinitions and descriptions of various humanphenotypic variations and this issue is freely avail-able online http://onlinelibrary.wiley.com/doi/10.1002/ajmg.a.v149a:1/issuetoc. Astandardized vocabulary for phenotypic varia-tions has been developed and is known as theHuman Phenotype Ontology (HPO). At presentthere are more than 11000 terms. The unifor-

mity of nomenclature helps in clinical diagnosticsbased on clinical features (Phenomyzer - http://compbio.charite.de/phenomizer/) and alsoresearch on relationships between clinical phe-notyping and molecular, cellular and biochemicalabnormalities and pathways.

Search for syndromic diagnosisOnce the clinical assessment of phenotypic varia-tions is done, the next step is to search for theetiological diagnosis or syndrome. There are twoprocesses for the diagnosis of a syndrome. Thereare some syndromes with characteristic pheno-types and especially the common syndromes canbe diagnosed based on the typical dysmorphicfeatures (gestalt). The key features, which arediagnostic clues for such syndromes, are knownas ‘Pearls of dysmorphology’. Figure 3 showstypical cases of some common syndromes withmalformations.

Use of Databases for Syndrome SearchMore than 5000 multiple malformation syndromesare described in literature and many new ones aregetting delineated. It is impossible to rememberall the features of each syndrome and to clinicallydiagnose the rarer conditions, even for a dys-morphology expert. For this purpose, there aredatabases of multiple malformation syndromes(Table 4) and these can help experts and newcom-

Table 3 Points to be noted in the history in the clinical evaluation of a patient with congenitalmalformation(s).

Prenatal history Drugs, infection, exposure to radiationMaternal illnesses like diabetes mellitus, epilepsyPrenatal ultrasonogram recordsAntenatal complications like polyhydramnios/ oligohydramnios

Family history A three-generation pedigree to note relatives with similarmalformations /clinical problemsAge of motherConsanguinityRecurrent abortions, stillbirths

Present / past history Growth and developmentSymptoms related to malformations of other systems e. g.convulsions, dyspnea, visual or hearing problemsSymptoms to suggest possibility of dysplasia or inborn errors ofmetabolism like progressive course, changing pattern ofdysmorphism etc.


http://onlinelibrary.wiley.com/doi/10.1002/ajmg.a.v149a:1/issuetoc

http://onlinelibrary.wiley.com/doi/10.1002/ajmg.a.v149a:1/issuetoc

http://compbio.charite.de/phenomizer/

http://compbio.charite.de/phenomizer/

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a) b)

c) d) e)

Figure 3 [a] Noonan syndrome – note upturned nose, ptosis, webbing of neck, pectus excavatumin lower part along with scar following operation for cardiac anomaly; [b] Rubinstein-Taybisyndrome with characteristic overhanging columella, grimacing smile and broad thumbs; [c]Coffin- Siris syndrome – hypoplastic terminal phalanx and nail of fifth finger and increasedbody hair; [d] Cornelia de Lange syndrome – note arched eyebrows meeting in the midlinewith an upturned nose; [e] A fetus with Cornelia de Lange syndrome with severe upper limbinvolvement in the form of ectrodactyly. Note the similarity of facial features with the face ofthe child in Figure 3[d].

ers in dysmorphology to get differential diagnosesfor each case. The phenotypic abnormalities whichare used as search handles in any database haveto be specific and uncommon. Eg. midlinecleft lip, preaxial polydactyly, microphthalmia aregood search handles while microcephaly, cleft lip,intellectual disability, short stature, hypertelorismare poor handles as they will give a long list ofdifferential diagnoses.

As medical genetics is a very rapidly advancingspecialty, literature search for latest developmentsshould be a part of syndrome diagnosis and geneticcounseling.

Role of Investigations in DysmorphologyDiagnosisInvestigations have two important roles in the eval-uation of a child with dysmorphism. The first is tolook for internal anomalies or associated treatableentities and surveillance for complications. Table5 gives some representative examples. In somecases the presence of internal malformations also

provides support to the clinical diagnosis as is thecase in Marfan syndrome.

The other important role of investigations inevaluation of a case with dysmorphology is identi-fication or confirmation of etiology.

Figure 4 Traditional karyotype showing trisomy21 due to translocation of an extra copyof chromosome 21 to chromosome 14.


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Table 4 List of databases that help in syndrome diagnosis.

Database Source FeaturesBaraitser-Winter DysmorphologyDatabase (LDDB)Winter-Baraitser NeurogeneticsDatabase (LNDB)

London Medical Databases (LMD)http://www.lmdatabases.com/

- Gives pictures; user friendly;needs to be purchased- Online version available -FACE2GENE LMD application

Pictures of Standard Syndromesand Undiagnosed Malformations(POSSUM)

http://www.possum.net.au/ Gives pictures; user friendly;needs to be purchased

Online Mendelian Inheritance inMan (OMIM)

http://omim.org/Provides detailed informationabout phenotypes as well asmutations

A catalogue of all genes andgenetic disorders; updated,comprehensive informationwith clinical synopsis andexternal useful links

Phenomyzer http://compbio.charite.de/phenomizer/

Uses Human PhenotypeOntology terms

Table 5 Representative examples of investigations suggested for some malformations or syndromes.

Malformation / Syndrome Associated malformation /problem

Investigations

Turner syndrome Cardiac anomalies, deafness,hypothyroidism, horseshoekidney

Echocardiogram,ultrasonography, thyroidhormone profile, audiometry

Congenital absence ofdepressor anguli oris

Cardiac anomalies Echocardiogram

Ear tag Deafness Evaluation of hearingMarfan syndrome Aortic root dilatation,

dislocation of lensEchocardiogram,Ophthalmologic evaluation

Bardet-Biedl syndrome Retinitis pigmentosa, deafness,renal problems

Hearing and ophthalmologicevaluation, renal function tests

Vertebral segmentation and ribcage abnormalities

Restrictive lung disease Pulmonary function tests

Genetic TestsGenetic disorders are classified into chromosomaldisorders, single gene (also known as Mendelian asthey follow Mendel’s laws of inheritance) disordersand multifactorial. There is no confirmatory lab-oratory investigation for multifactorial disordersbut chromosomal abnormalities may need to beruled out in some malformations before labelingthe malformation as multifactorial. Chromosomeshave been studied by traditional karyotyping formore than 5 decades (Figure 4). However, nowtechniques using DNA principle of complementaryregions (A to T and G to C) annealing each other areused to study small regions on chromosomes whichare beyond the resolution of traditional karyotyp-

ing. These molecular cytogenetic techniques areFluorescence In Situ Hybridization (FISH), Quan-titative Fluorescence Polymerase Chain Reaction(QF-PCR), Multiplex Ligation Probe Amplification(MLPA) and Cytogenetic MicroArray (CMA). Table6 gives principles and appropriate indications ofthese tests (Figure 5).

The other group of tests is for identifyingmutations in genes causing monogenic disorders.Most of the mutations are variations in the DNAsequence and are identified by Sanger sequencingusing automated sequencers (Figure 6). Before or-dering the test the clinician should have a workingdiagnosis with a known causative gene. It needs tobe noted that the identification of a causative geneconfirms the diagnosis; absence of mutation in a


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suspected gene does not rule out the diagnosis.The causes in such situation can be different like,inability of the technique to detect the type of mu-tation, mutation in other parts of gene like introns,promoter, etc. which are not usually tested, orthe causative gene may be other than the onetested. It has been seen in a number of disordersthat different genes may cause a clinically similarphenotype and some of the genes still may nothave been identified. The updated informationabout all known genes and phenotypes is availableon Online Mendelian Inheritance in Man (OMIM), afree database.

a) b)

c) d)

Figure 5 [a] Chromosome 5p microdeletion syn-drome –also known as Cri-du-chat syndrome [b]FISH with probes for terminal region of chromo-some 5 showing only one copy confirming thedeletion on the other copy of 5p [c] Williamssyndrome showing mild dysmorphism but withcharacteristic face – a case for gestalt diagnosis [d]MLPA showing decreased ratio (0.5) of probes onchromosome 7q11 region (patient’s DNA sampleto the DNA of a normal individual), while otherprobes show a normal ratio around 1.

For most of the Mendelian disorders, hundredsof mutations are identified and to identify themutation, sequencing of all coding regions (exons)needs to be done. As many genes have 20 or moreexons, the process is tedious and costly. The use ofSanger sequencing for diagnosis in clinical settingsgets complicated by involvement of multiple genescausing the clinically indistinguishable phenotypes.This problem has been solved by the latest highthroughput sequencing technique known as Next

Generation Sequencing (NGS). NGS is routinelyused in clinical settings to sequence multiple genesin one go (eg., genes in RAS- MAPK pathway whichcause clinically overlapping phenotypes like Noo-nan syndrome, Cardio-facio-cutaneous syndrome,Costello syndrome, etc). It is also useful forsequencing large genes like the FBN1 gene whichcauses Marfan syndrome. NGS based sequencingof coding regions (exons) of all genes known asWhole Exome Sequencing (WES) or Whole GenomeSequencing (WGS) is used in cases where identifi-cation of the candidate gene based on phenotypeis not possible as the phenotype is novel or sub-tle. This helps in identification of the causativemutation in 30 to 50% of cases with developmentaldisorders and causative genes for many novelphenotypes are getting identified. As is the casein CMA, WES identifies thousands of variations ineach individual and most of them are polymorphicand non-pathogenic. Dissecting the results ofWES to identify the causative pathogenic mutationis a challenging task which needs high level ofcomputational expertise, bioinformatic tools alongwith clinical correlation and functional studies ofthe variation. NGS based mutation detection incases with malformation with or without develop-mental disabilities helps to identify new mutationsand sometimes new genes, and also widens thephenotypic spectrum by identifying a mutation in aknown gene which was not suspected on the basisof the clinical phenotype.

Figure 6 [a] A fetus with Apert syndrome show-ing tower skull and syndactyly [b] CT scan ofthe head showing closed coronal sutures and wideopen sagittal suture [c] Sequencing results of a partof FGFR2 gene showing the p.Ser252Trp mutation.


GeNeViSTATable 6 Genetic investigations to study chromosomes and chromosomal disorders.

Technique /Investigation

Advantages Limitations Indication

Karyotype Can look at allchromosomes inone go and theclinical suspicion ofa specific diagnosisis not necessary

Level of resolution is lowand sub-microscopicabnormalities cannot bedetected.Needs living cells.Cell culture is needed for3 to 10 days.

Clinical diagnosis of achromosomal disorder Downsyndrome, Turner syndrome,Trisomy 13 or 18; a fetus withhydrops fetalis or any majormalformation; liveborn or astillborn with multiplemalformations; intellectualdisability; short stature in aprepubertal girl; disorder ofsex development

FISH Can detectsub-microscopicdeletions onchromosomes. Noneed of cell cultureor live cells.Short reportingtime of 1 or 2 days.

Clinical suspicion of aspecific disorder(s) isneeded, so that theprobes for theappropriate region canbe used.Can usually test 1 to 3regions in one test.

Rapid prenatal testing fortrisomy 21, 13 or 18;Microdeletion syndromes likeWilliams syndrome,Velo-cardio-facial syndromeetc.

QF PCR Does not need cellculture. Reportingtime one or 2 days

Can test up to 5chromosomes in one go.

Rapid prenatal diagnosis forcommon aneuploidies on fetalsample (amniotic fluid).

MLPA (Probe sets areavailable for knownmicrodeletion / duplicationsyndromes or for ends ofall chromosomes)

Does not need livecells or cell culture.Rapid like FISH butcan test 40 targetregions in one test.

Can test for regionsknown for syndromicetiology andincorporated in theprobe set used for MLPA.

Useful to test for many welldelineated microdeletionsyndromes in a child withintellectual disability.

CMA(Probes spanning thewhole genome)

Does not need livecells or cell culture.Covers the entiregenome. Has ahigh resolution.

Variants of uncertainsignificance identified

First tier of test for evaluationof a child with intellectualdisability / dysmorphism ifthere is no obvious cause onclinical evaluation.

Note: FISH: Fluorescence In Situ Hybridization; QF-PCR: Quantitative Fluorescence Polymerase Chain Reaction;MLPA: Multiplex Ligation Probe Amplification; CMA: Cytogenetic MicroArray

Genetic Counseling and Prenatal Diag-nosisCongenital malformations are heterogeneous andas a group, a common entity. Counseling regardingprognosis, treatment options, and outcome withsurgical and non-surgical interventions is impor-tant in prenatally as well as postnatally detectedmalformations. Genetic counseling about the riskof recurrence and prevention by prenatal diag-nosis during the next pregnancy is an importantpart of management of a family with a child ora pregnancy with birth defect. Accurate geneticcounseling needs correct etiological diagnosis ofthe proband (child or fetus with birth defect). Care-

ful ultrasonographic evaluation of fetus at 18 to20 weeks can prenatally detect most of the majormalformations. Fetal autopsy has a major role inthe evaluation of stillbirths and fetuses terminatedafter prenatal diagnosis of a malformation.

ConclusionEvaluation of an individual with dysmorphismneeds expertise and knowledge about syndromes,basic genetics and principles of genetic investiga-tions and their interpretation. Etiological diagnosishelps in appropriate management of the affectedindividual as well as in accurate genetic counselingof the family.


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GeNeViSTA Genetic Approach to Congenital Malformationsiamg.in/genetic_clinics/adm/articlepdf/July-September-2018-04-gene... · Table 3 Points to be noted in the history in the clinical