Volume 14 | Issue 3 | July - September 2021 Genetic

Volume 14 | Issue 3 | July - September 2021

ISSN : 2454-8774

Table of Contents

Official Publication of Society for Indian Academy of Medical Genetics

GeneticClinics

Office Bearers of SIAMG

Editor

Assistant Editors

Associate Editor

Shubha R Phadke Prajnya Ranganath

Ashwin Dalal, Girisha KM, Dhanya Lakshmi N

Patron

IC Verma

President

Ashwin Dalal

Secretary

Ratna Dua Puri

Treasurer

Shagun Aggarwal

Address for correspondenceDr Shubha R Phadke

Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow-226 014, EPABX: 0522-2668005-8 | Phone: 0522 249 4325, 4342 | E-mail: [email protected]

Page 01

Deciphering Clues to Genotype-Phenotype Correlation

Methylation, Monogenic Disordersand More

Challenges of Molecular Analysis ofCongenital Adrenal Hyperplasia CausedDue to Steroid 21 Hydroxylase Deficiency

Exome Sequencing Reveals a NovelHomozygous Variant in WDR62 Gene ina Family with Primary Microcephaly

PhotoQuiz - 53

Genetic Counseling of PrenatallyDetected Sex Chromosome Anomalies

GeNeDit

GeNeXprESS

GeNeViSTA

Clinical Vignette

PhotoQuiz

GeNeViSTA

Page 02

Page 06

Page 14

Page 20

Cover page

PhotoQuiz - 53Contributed by: Dr Jai Prakash Soni

Correspondence to: Dr Jai Prakash Soni. Email: [email protected]

This baby, delivered at 20 weeks of gestation, was noted to have significant

dysmorphism with joint contractures. The clinical photographs and whole body skeletal

radiographs (anteroposterior and lateral views) are provided. Identify the condition.

Please send your responses to [email protected]

Or go to http://iamg.in/genetic_clinics/photoquiz_answers.phpto submit your answer.

Department of Pediatrics, Dr Sampurnanand Medical College (SNMC), Jodhpur, Rajasthan, India

Answer to PhotoQuiz 52

Correct responses were given by:

Dyggve-Melchior-Clausen (DMC) disease is an autosomal recessive skeletal dysplasia caused by biallelic mutations in the DYM gene (OMIM*607461). It is characterized by disproportionate short stature, microcephaly, facial dysmorphism, psychomotor delay and intellectual disability. Skeletal radiographs in patients with DMC disease show features of spondyloepimetaphyseal dysplasia along with additional typical findings such as small iliac wings with lacy appearance of the iliac crests, platyspondyly with notched end plates, and multicentric ossification of proximal humeral and femoral epiphyses. Smith-McCort dysplasia 1 is an allelic disorder, with similar skeletal findings, but with normal intellect.

Dyggve-Melchior-Clausen disease (OMIM #223800)

1. Dr Poonam Singh Gambhir, Vardaan Genetic and Diagnostic Centre, Kanpur

2. Dr Kruti Varshney, Centre for Human Genetics, Bangalore

3. Dr Anupriya Kaur, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh

4. Dr Komal Uppal, All India Institute of Medical Sciences (AIIMS), New Delhi

5. Dr Moni T Bhatia, Noble Heart and Superspeciality Hospital, Rohtak

6. Dr Vibha Jain, Anuvanshiki - The Genetic Centre, Ghaziabad

7. Dr SG Vijayasri, Institute of Child Health and Hospital for Children, Chennai

8. Dr Ashka Prajapati, Genetic Care Clinic and CIMS Hospital, Ahmedabad

9. Dr Haseena A, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow

10. Dr Lekshmi S Nair, NIMS Medicity, Neyyatinkara, Kerala

11. Dr Bhawana Aggarwal, All India Institute of Medical Sciences (AIIMS), New Delhi

12. Dr Beena Suresh, Mediscan Systems, Chennai

13. Dr Surya G Krishnan, Indira Gandhi Institute of Child Health, Bangalore

14. Dr Meenakshi Lallar, Prime Prenatal Imaging and Diagnostics, Chandigarh & Medgenome, Bengaluru

GeNeDit

Deciphering Clues to Genotype-Phenotype CorrelationEditorial

The beginning of the era of mutation detectionin clinical practice was very exciting. Some‘lumped’ phenotypes got separated while somedisorders with great phenotypic similarities wereeither found to be allelic or disorders sharinga common pathway. One gene and multiplephenotypes was found to be not uncommon.Incomplete penetrance and variable expressiondid not remain concepts and observations buthad molecular evidence to support them.Intrafamilial variability more common withautosomal dominant disorders but also seen inautosomal recessive disorders led to the searchfor modifier genes. Interaction between alpha andbeta globin genes provided some insights into thegenotype-phenotype correlations of thalassemiaintermedia. Null alleles, specific locationsof mutations and gain-of-function mutationsprovided some genotype-phenotype correlationsin beta thalassemia, osteogenesis imperfecta, etc.Specific single causative mutation responsible forthe disease in concern has been observed invery few disorders such as sickle cell disease,type V osteogenesis imperfecta, and Caffeydisease. In certain others such as achondroplasia,Apert syndrome and Hutchinson-Gilford progeriasyndrome, one or a few mutations have beenfound to account for majority of the cases. Somecorrelation based on the nature and position ofthe mutation is understood, such as out-of-frame/frameshifting deletions in the dystrophin geneleading to the more severe phenotype ofDuchenne muscular dystrophy versus the in-framedeletions causing the less severe phenotype ofBecker muscular dystrophy. However, this alonecannot explain the phenotypic variation in allcases. In general, for most of the disorders nogenotype-phenotype correlation is observed.

Genetic heterogeneity and phenotypicheterogeneity are challenges in clinical practice.Next generation sequencing-based diagnosticshave provided solutions to some extent to thegenetic heterogeneity. However, prediction ofphenotype continues to remain a big questioneven for known pathogenic variations. One of themost important causes of marked phenotypicvariability observed in females with fragile Xsyndrome is lyonization leading to mosaicism forthe mutated and fully methylated allele of FMR1

gene. Mosaicism for number of repeats andmosaicism for methylation of FMR1 promoter hasbeen observed in males. The methylation status ofFMR1 gene promoter has shown correlation withFMR1mRNA and neurodevelopmental dysfunction.The GenExpress of this issue discusses the useof methylation of FMR1 gene promoter insamples of newborn screening, for diagnosisand prognostication. Many genes involved inchromatin modelling influence the expression ofmany other genes, and mutations in these genesthus cause phenotypic abnormality due to changesin the expression of genes under their control. Themodification of methylation of many genes in thegenome by pathogenic sequence variations ingenes for monogenic syndromes like Coffin-Sirissyndrome, Rubinstein-Taybi syndrome, etc. hasbeen reported in recent literature. Research inthis area has successfully provided specificmethylation signatures of these monogenicdisorders. Studies of correlation of the expressionof genes with modified methylation, sequencevariation in concern and the phenotypes, will beuseful in classifying novel sequence variationsas pathogenic or non-pathogenic, and alsomay provide insights into genotype-phenotypecorrelations. The GenExpress in this issuealso mentions another interesting study ondifferentially methylated regions (DMRs) thatundergo demethylation in late gestational age incord blood cells, which can be used to correctlyassess the gestational age of a neonate.

As next-generation sequencing is coming intoclinical practice for population-based screeningfor carriers of recessive disorders and fornewborn screening for early-onset serious geneticdisorders, there is a strong need to find outgenetic modifiers so as to go into the depthof genotype-phenotype correlation. Not onlysequence variations in the genome and genes ofthe pathway or protein complexes but the variousepigenetic mechanisms affecting gene expressionmay provide clues to the unanswered questions ofgenotype-phenotype correlations.

Dr. Shubha Phadke1st July, 2021

Genetic Clinics 2021 | July - September | Vol 14 | Issue 3 1

Clinical Vignette

Exome Sequencing Reveals a Novel Homozygous Variant inWDR62 Gene in a Family with Primary Microcephaly

Ikrormi Rungsung1, Mahesh Kamate2, Ashwin Dalal11Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

2Department of Pediatric Neurology, Jawaharlal Nehru Medical College, KLE University, Belgaum, Karnataka, India

Correspondence to: Dr Ashwin Dalal Email: [email protected]

Abstract

Autosomal recessive primary microcephaly 2(MCPH2) is a neurodevelopmental disease thatcauses reduction in brain size. Homozygous orcompound heterozygous mutations in the WDR62gene, located at the chr19q13.12 locus arereported to result in MCPH2. The most commonfeatures are reduced skull circumference andintellectual disability with or without corticalmalformations. We describe a genetic variant intwo siblings, a 4-year-old boy and a 15-month-oldgirl, with congenital microcephaly, globaldevelopmental delay, intellectual disability andhyperactivity. Exome sequencing was performedon the genomic DNA and analyses revealeda novel frameshift deletion, NM_001083961.2;c.669delC; p. Phe223fs in exon 6 of the WDR62gene. The variant c.669delC causes a frameshiftat p. Phe223fs position of the WD40-repeat 62protein (WDR62) protein and is classified as a‘pathogenic’ variant according to the AmericanCollege of Medical Genetics/ Association forMolecular Pathology (ACMG/AMP) classification.The unaffected parents were found to beheterozygous for this mutation. Our findingsexpand the mutation spectrum of WDR62gene-related phenotype.

Introduction

The worldwide incidence of microcephaly variesfrom 1.3 to 150 per 100,000 populations (Tolmieet al., 1987). Microcephaly has been reportedmore commonly in Asians and Arabs dueto consanguineous unions (Hussain & Bittles,1998; Thornton & Woods, 2009; Woods et al.,2005). Autosomal recessive primary microcephaly

(MCPH) is characterized by a small headcircumference ranging from 2 standard deviations(SD) to 11 SD below the mean for age andsex-matched individuals. The affected patientsshow delayed psychomotor development and mildto severe intellectual disability, which is oftenaccompanied by other brain malformations. TheOnline Mendelian Inheritance in Man (OMIM) hasreported twenty-seven loci or genes for primarymicrocephaly. It has been reported that mostof the MCPH-associated gene products arecentrosomal proteins and play diverse rolesduring neurogenic mitosis (Cox et al., 2006).Here we report a novel homozygous frameshiftvariant in WDR62 gene in two siblings bornto consanguineous parents, identified throughexome sequencing. WDR62 gene is expressed inthe neuroepithelium of apical precursors duringmitosis (Nicholas et al., 2010).

Patient details

The proband is a 4-year-old boy, born at termgestation, with a birth weight of 3.5 kg. He wasnoted to have small head size at birth. Thereis second degree consanguinity in the parents(Figure 3A). Antenatal and perinatal periods wereuneventful. There was global developmental delaywith sitting attained at 10 months and walking at 2years. He spoke his first word at 3 years and haddrooling of saliva from 2 years of age. He was firstseen at 15 months of age and was on follow-upthereafter. At the time of initial examination,the head circumference was 37.0 cm (z-score−7.5), height was 73 cm (z-score −3) and weightwas 7.5 kg (z-score −3.3). Head circumferenceat 3.5 yrs was 39.0 cm (z score −6.7), heightwas 87 cm (z-score −3.3) and weight was 8.0 kg


Clinical Vignette

(z-score −4.8). Currently patient has hyperactivityand aggressiveness. He has left hand preference.There was no history of seizures. MRI brain wasnormal with simplified gyral pattern. There was noventriculomegaly, lissencephaly, polymicrogyria orcerebellar dysplasia. The thickness of the cortexwas also normal. The second sibling, a femalechild, who is now 15 months of age, weighed 2.0 kgat birth and was also noted to have a small-sizedhead. She also has global developmental delay(attained head control at 4 months; but cannotsit or stand and has not attained languagemilestones), and her head circumference was36.5 cm (z score −6.6), height was 63 cm (z-score−3.3) and weight was 5.7 kg (z-score −3.8) at10 months of age. She had drooling of salivawith normal tone and no seizures. Both siblingsshowed mild dysmorphic features in the form oftriangular facies, broad nasal root, bulbous tip ofnose, smooth philtrum, thin upper lip and thicklower lip, and smooth philtrum (Figure 1).

Figure 1 Photographs of proband and siblingshowing microcephaly.

Genomic DNA isolation

We collected 2 milliliter of peripheral blood samplein EDTA vacutainer tube (BD-Plymouth, PL6 7BP,UK) from the proband, his sibling and bothparents, after obtaining informed consent. Totalgenomic DNA was isolated from blood usingthe HiGEnoMB DNA purification kit (HiMediaLaboratories, LLC).

Whole-exome sequencing

Exome sequencing was performed on genomicDNA. The SureSelect Clinical Research Exome V2

kit (Agilent SureSelect technology) was used tocapture and enrich regions from exons alongwith 75,000 splice sites of non-coding exons,more than 12,000 deep intronic sites andover 800 promoter regions. The captured andenriched library was amplified and sequenced onthe Illumina sequencer, for 100X coverage. Thereads were assessed for quality control using theFastQC and mapped to the human referencegenome 19 (hg19/GRCh37) using the BWA MEMprogram. The variant calling was done using GATKhaplotypecaller and the VCF file was annotatedagainst the genomic variation populationdatabases and bioinformatics prediction tools. Thepopulation databases used were 1000 Genomes,gnomAD, Exome Variant Server, GenomeAsia, inhouse databases and the bioinformatics predictiontools used were MutationTaster and CombinedAnnotation Depletion Dependent (CADD).

Sanger sequencing

Specific primers F1 and R1 were designedfor the WDR62 gene mutation and amplified.PCR products were visualized in 2% agarosegel electrophoresis and then sequenced on ABIPrism A3730-automated sequencer (PE AppliedBiosystems, Thermo Fisher Scientific, Waltham MA,USA). The Sanger sequence chromatograms werevisualized with FinchTV (Geospiza, Inc. Seattle, WA,USA) for the presence or absence of the mutation.

Results

Exome sequencing was performed on theproband which revealed 131,974 total variants. Toidentify the causative variant, the polymorphicvariants [with minor allele frequency (MAF) ≥0.01] present in the 1000 Genomes, ExAC,EVS, gnomAD, GME, cg69 and in-house exomedatabases, were excluded. Further, we lookedfor variants in the exonic regions and splicesites. This led to 22 non-synonymous variants, 2stop-gain variants, 2 frameshift deletions and oneframeshift insertion variant in homozygous state(Figure 2). A novel frameshift deletion variant(NM_001083961.2; c.669delC) in the WDR62 (OMIMID #604317 (https://omim.org/) gene waschosen as the candidate variant, because thereported WDR62-associated phenotype matchedthe proband’s clinical features. The variant wassubmitted to ClinVar with the accession numberVCV000818086.1. It is classified as ‘pathogenic’


https://omim.org/

Clinical Vignette

as per the ACMG/AMP guidelines (Figure 3B).WDR62 gene mutations are known to causeautosomal recessive primary microcephaly 2, withor without cortical malformations (MCPH2). Insilico prediction tools showed the variant to bedisease-causing. Sanger sequencing confirmed thehomozygous single base pair deletion in exon 6 atNM_001083961.2; c.669delC mutation in WDR62gene in both the proband and the sibling, and thesame was found to be present in heterozygousform in both the unaffected consanguineousparents (Figure 3C).

Figure 2 Filtering strategy of exome sequencingdata.

Discussion

Microcephaly-2 with or without corticalmalformations is inherited in an autosomalrecessive fashion and shows significant phenotypicvariability. Patients with pathogenic variants inWDR62 have head circumferences ranging fromlow-normal to severe (−9.8 SD) microcephaly, andmost patients reveal various types of corticalmalformations in brain MRI. All patients havedelayed psychomotor development but seizuresare variable.

We have presented genetic evidence of anovel frameshift deletion in WDR62 gene linkingto autosomal recessive primary microcephaly2 (MCPH2). Our data revealed that thisframeshift deletion co-segregated with the diseasephenotype, since it was present in the affectedsibling in the homozygous state and in theheterozygous state in both parents. In humans,WDR62 gene encodes for WD40-repeat protein 62

(WDR62) protein, which has 1518 amino acids andcontains tryptophan-aspartic acid (WD) dipeptiderepeats. Studies have shown that WDR62 proteinbinds with the centrosomal protein CEP170 and itstabilizes the mitotic spindle during metaphase. Itis also known that the WDR62 protein plays a rolein neurogenesis via the c-Jun N-terminal kinase(JNK) signaling pathway (Bhat et al., 2011).

There are reports of consanguineous familieswith microcephaly-2 with cortical malformations,including polymicrogyria, schizencephaly, andsubcortical heterotopia. Another study from Indiareported on 2 different homozygous truncatingWDR62 mutations in unrelated consanguineousfamilies with MCPH2 with cortical malformations(Bhat et al., 2011). Our patient did not have anybrain malformations other than simplified gyralpattern.

The WDR62 gene has 32 exons and differenttypes of mutation like missense, nonsense, splicesite and indels have been reported across theexonic regions (Figure 3D). Bilguvar et al haveidentified homozygosity for a 4-basepair deletion(TGCC) in exon 31 of the WDR62 gene at codon1402, G-to-A substitution in exon 12 at codon 526,G-to-C transversion in exon 6 at codon 224, C-to-Ttransition in exon 11 at codon 470, and a 17-bpdeletion in exon 30 at codon 1280 (Bilgüvar et al.,2017). Homozygous 1313G-A transition in exon 10and duplication 4241dupT in exon 31 of theWDR62 gene were also reported by another group(Roberts et al., 1999). Yu et al have also reportedhomozygous 1531G-A transition in exon 11, 1-bpinsertion (3936insC) in exon 30, 1-bp deletion(363delT) in exon 4 of the WDR62 gene and a193G-A transition in exon 2 of the WDR62 gene (Yuet al., 2010). In addition, a 1-bp deletion (2083delA)in exon 17 and a 2-bp deletion at 2472_2473delAGin exon 23 of WDR62 gene have also been reported(Nicholas et al., 2010).

Our results assert that NM_001083961.2;c.669delC variant in WDR62 gene explainsthe clinical features of microcephaly, globaldevelopmental delay, intellectual disability andhyperactivity observed in the present sibship andexpands the genotypic spectrum of variants in theWDR62 gene.

Web resources• OMIM, https://www.omim.org/• MutationTaster,http://www.mutationtaster.org/


https://www.omim.org/

http://www.mutationtaster.org/

Clinical Vignette

Figure 3 A. Pedigree of the family. B. Variant visualization using Integrated genome viewer for c.669delCvariant in exon 6 of the WDR62 gene. C. Segregation analysis using targeted Sanger sequencingin the patient’s family. D. Schematic representation of all WDR62 exons (adapted from theEnsembl browser) showing exon-wise known mutations with our novel variant in red(NM_001083961.2; c.669delC) for transcript ID ENST00000401500.7.• CADD,

http://cadd.gs.washington.edu/score• 1000 Genomes Project,http://phase3browser.1000genomes.org/index.html• gnomAD,http://gnomad.broadinstitute.org/• GenomeAsiahttps://genomeasia100k.org/• Exome Variant Serverhttps://evs.gs.washington.edu/EVS/

References

1. Bhat V, et al. Mutations in WDR62, encoding acentrosomal and nuclear protein, in Indianprimary microcephaly families with corticalmalformations. Clin Genet. 2011; 80: 532–540.

2. Bilgüvar K, et al. Whole exome sequencingidentifies recessive WDR62 mutations insevere brain malformations Nature. 2010;467:207–210.

3. Cox J, et al. What primary microcephaly can tellus about brain growth. Trends Mol Med. 2006;12: 358–366.

4. Hussain R, et al. The prevalence anddemographic characteristics of consanguineousmarriages in Pakistan. J Biosoc Sci. 1998; 30:261–275.

5. Nicholas AK, et al. WDR62 is associated withthe spindle pole and is mutated in humanmicrocephaly. Nat Genet. 2010; 42: 1010–1014.

6. Roberts E, et al. The second locus for autosomalrecessive primary microcephaly (MCPH2) mapsto chromosome 19q13.1-13.2. Eur J Hum Genet.1999; 7: 815–820.

7. Thornton GK, et al. Primary microcephaly: do allroads lead to Rome? Trends Genet. 2009; 25:501–510.

8. Tolmie JL, et al. Microcephaly: Geneticcounselling and antenatal diagnosis after thebirth of an affected child. Am J Med Genet.1987; 27: 583–594.

9. Woods CG, et al. Autosomal recessive primarymicrocephaly (MCPH): A review of clinical,molecular, and evolutionary findings. Am J MedGenet. 2005; 76: 717–728.

10. Yu TW, et al. Mutations in WDR62,encoding a centrosome-associated protein,cause microcephaly with simplified gyri andabnormal cortical architecture. Nat Genet.2010; 42: 1015–1020.


http://cadd.gs.washington.edu/score

http://phase3browser.1000genomes.org/index.html

http://phase3browser.1000genomes.org/index.html

http://gnomad.broadinstitute.org/

https://genomeasia100k.org/

https://evs.gs.washington.edu/EVS/

GeNeViSTA

Challenges of Molecular Analysis of Congenital AdrenalHyperplasia Caused Due to Steroid 21 Hydroxylase Deficiency

Sudhisha Dubey1, Renu Saxena1, Vinu Narayan2, Ratna Dua Puri1, Ishwar C Verma11Institute of Medical Genetics and Genomics, Sir Ganga Ram Hospital, Rajinder Nagar, New Delhi, India

2Rainbow Children’s Hospital, Marathahalli, Bengaluru, Karnataka, India

Correspondence to: Dr Sudhisha Dubey Email: [email protected]

Abstract

Congenital adrenal hyperplasia (CAH) due to21-hydroxylase deficiency is an autosomalrecessive disorder which results from inheriteddefects in the steroid 21-hydroxylase enzymeencoded by the CYP21A2 gene. Molecular analysisof CYP21A2 is important for confirming thediagnosis, carrier screening, providing accurategenetic counseling, and calculating risk ofrecurrence in each pregnancy. An interestingfeature of the CYP21A2 gene is its location inthe variable genomic regions called RCCX andpresence of its highly homologous CYP21A1Ppseudogene that makes molecular analysis quitechallenging as compared to other monogenicdisorders. Here we discuss the complexity ofthe CYP21A2 gene and the importance ofcomprehensive molecular analysis of CYP21A2 foraccurate interpretation of the results citingmolecular analysis of two interesting CAH cases.

Keywords: Congenital adrenal hyperplasia,CYP21A2 gene, CYP21A1P, pseudogene, variants,MLPA, deletion, duplication

Introduction

Congenital adrenal hyperplasia (CAH) dueto 21-hydroxylase deficiency (OMIM# 201910),is an autosomal recessive disordercaused by inherited deficiency of steroid21-hydroxylase (21OH) enzyme in thesteroid biosynthesis pathway in the adrenalcortex. 21OH enzyme acts on progesteroneand 17-hydroxyprogesterone (17OHP) andconverts these to deoxycortisosterone and11-beta-hydroxylase respectively, which arefurther converted into aldosterone and cortisol by

other enzymes in the steroidogenic pathway.Deficiency of 21OH enzyme results in shuntingof 17OHP and progesterone into the adrenalpathway resulting in excessive production ofandrogens and deficiency of aldosterone andcortisol (Figure 1). Excessive androgens lead toprenatal virilization in females and rapid somaticgrowth in both sexes (White & Speiser, 2000).Deficient cortisol level disrupts the negativefeedback to the anterior pituitary that resultsin constant secretion of adrenocorticotropichormone (ACTH) that overstimulates the adrenalcortex to secret more of cortisol. Due to 21OHdeficiency in the adrenal pathway, the cortisol isnot secreted and adrenals become hyperplasticdue to overstimulation of ACTH in fetal life.That is how this condition obtained its name as“congenital adrenal hyperplasia”.

CAH is divided into classic and non-classic (NC)CAH. Classic CAH is again divided into salt-wasting(SW) and simple virilizing (SV) forms. SW-CAH is asevere form characterised by deficiency of bothcortisol and aldosterone and found in about 75%of patients. Aldosterone deficiency predisposesSW-CAH patients to develop hyponatremicdehydration which is fatal if not treated withglucocortcoids in time. SV-CAH is a milder formfound in about 25% of CAH patients. Aldosteronelevels are adequate to maintain sodium balancein the SV form and hence there is normallyno salt wasting. The NC form is asymptomaticat birth and presents with various degrees oflate-onset hyperandrogenism (White & Speiser,2000). Prenatal virilisation may or may not bepresent in the mild NC form but is always presentin the SW or SV classic forms.

The overall incidence of CAH in the generalpopulation worldwide is between 1 in 10,000 to 1in 20,000 live births for the classic form of CAH


GeNeViSTA

Figure 1 Steroid pathways for biosynthesis of progesterone, aldosterone, cortisol, androgens(testosterone and dihydrotestosterone), and estrogens (estradiol) are arranged from left toright. The enzymatic activities catalyzing each bioconversion are written in boxes. For thoseactivities mediated by specific cytochromes P450, the systematic name of the enzyme (“CYP”followed by a number) is listed in parentheses. CYP11B2 and CYP17 have multiple activities.The planar structures of cholesterol, aldosterone, cortisol, dihydrotestosterone, and estradiolare placed near the corresponding labels (adapted from White & Speiser, 2000).

(Therrell et al., 2001). However, the prevalenceof classic CAH in India is 1 in 5762 accordingto a recent survey (ICMR task force, 2018).Non-classic CAH is one of the most commonautosomal recessive disorders in humans andaffects approximately 1 in 1,000 individuals(Speiser et al., 1985).

Steroid 21OH enzyme, is encoded by theCYP21A2 gene located on chromosome 6 (6p21.3)in the HLA class III of the major histocompatibility(MHC) region (Yang et al., 1999). About 30 kbupstream a non-functional pseudogene CYP21A1Pis located that shares about 98% sequencehomology to CYP21A2. About 95% of thepathogenic variants are pseudogene derived andare transferred from CYP21A1P to CYP21A2 bygene conversion events (Higashi at al., 1986).The remaining 5% are new/rare and unique forsingle families or considered as population specific(White & Speiser, 2000; Stikkelbroeck et al., 2003).A compilation of 233 pathogenic variants and theirclinical classification have been done recently(Concolino & Costella, 2018).

CYP21A2 gene is a part of the genetic unitcomprising of RP2-C4B-CYP21A2-TNXB genes knownas the RCCX module. Each chromosome bears twoRCCX modules; one with the functional CYP21A2gene and other with the non-functional CYP21A1P

as shown in Figure 2. Majority of the individualshave a bimodular haplotype i.e., two modulespresent on each chromosome. However, threemodules have also been reported to be presenton one chromosome which is known as thetrimodular haplotype. In the trimodular haplotypeeither two CYP21A1P and one CYP21A2 or oneCYP21A1P and two CYP21A2 are present on onechromosome (Figure 2). The later has two copiesof functional gene on a chromosome resulting induplication of the CYP21A2 gene that complicatesthe molecular analysis of the CYP21A2 gene.

In about 20-30% of cases, the large 30kbdeletion extends from somewhere between exon3 of CYP21A1P through C4B to the correspondingpoint in CYP21A2 yielding a single copy with 5´ endof CYP21A1P and 3´ end of CYP21A2, also knownas the chimeric gene. Nine different chimerashave been reported depending on the extent ofdeletion involved (Chen et al., 2012). Extent of thedeletion also helps in determining the genotype-phenotype correlation (Narasimhan et al., 2019).

Materials and methods

Written informed consent was obtained fromthe parents of both patients. About 100 ng ofeach genomic DNA was subjected to selective


GeNeViSTA

Figure 2 Schematic diagram of the organization of the RCCX modules. The most common is thebimodular haplotype with two RCCX modules, one with pseudogene CYP21A1P and otherfunctional CYP21A2 gene. Trimodular haplotype with three RCCX modules can result induplication of the CYP21A2 gene. C4 (C4A and C4B) gene encodes the fourth component of theserum complement. RP2, a truncated copy of RP1, encodes the threonine kinase enzyme andTNXB encodes tenascin-X an extracellular matrix protein. TNXA is a non-functional homologueof the TNXB gene (adapted from Sweeten et al., 2008).

Figure 3 A. PCR amplification of the CYP21A2 gene into two fragments; fragment A (1130bp) andfragment B (2127bp). M- DNA Ladder; Lanes 2 & 6-Fragment A; Lanes 3 & 7 – Fragment B; Lane4-5, 8-9 – Absence of bands or amplification indicating gene deletion. B. Purified PCR productsof fragment A and B with MassRuler (MR). (Dubey et al., 2017)

amplification of CYP21A2 into two large fragmentswith two sets of primers highly specific to theactive i.e., CYP21A2 gene (Figure 3A). Absence ofbands indicate the deletion of 8 bp of exon 3 orwhole of the active gene which is confirmed byMLPA. These fragments were purified using theQiagen kit (QIAamp PCR Clean-up, Qiagen GmbH,Hilden, Germany) and quantified with MassRuler

(Fermentas Life Sciences, Thermo Fisher Scientific,Waltham MA, USA) (Figure 3B) (Dubey et al.,2017). Purified products were subjected to directsequencing using ABI 3500 Genetic Analyser (PEApplied Biosystems, Thermo Fisher Scientific,Waltham MA, USA). Pathogenic variants werescreened using Chromas v2.4 and SeqScapev2.1.1 (Applied Biosystems) against the NCBI


GeNeViSTA

Figure 4 Partial electropherogram showing homozygous I2g (c.293-13A/C>G) mutation detected in theproband (Patient 1).

reference sequence NM_000500 and transcript IDENST00000418967. Multiple ligation dependentprobe amplification (MLPA) was done using SalsaMLPA Kit P050-C1 (MRC-Holland, Amsterdam, TheNetherlands) to detect deletions and duplications.

Patient description and results

Patient 1: A five-years-old female child presentedwith ambiguous genitalia at birth. She hadcomplete labial fusion and clitoral hypertrophy.Her karyotype was normal female (46, XX) andultrasound-abdomen revealed bilateral ovaries.She had elevated levels of 17 OHP (greater than 37ng/mL), renin (greater than 500 ng/mL/hour),potassium (7.8 mEq/L) and low level of sodium(116 mEq/L). She was reported to have sevenmutations i.e., I2g (c.293-13A/C>G) (intron2),c.332_339delGAGACTAC (exon 3), c.515T>A (exon4), c.710T>A (exon 6), c.713T>A (exon 6), c.719T>A(exon 6), and c.923_924insT (exon 7) by NGS. Allmutations were in heterozygous form exceptsplice site mutation I2g (c.293-13A/C>G) in intron 2of the CYP21A2 gene. Snapshots of Integrativegenome viewer (IGV) software and MLPA ratiochart were also provided that clearly illustratedpresence of these mutations.

The proband was referred to us for validationand segregation of pathogenic variants in herparents, her paternal aunt and the aunt’shusband, as her aunt was pregnant and the familywanted prenatal diagnosis (PND) to be done.Sequencing of the proband was carried out tovalidate the seven reported pathogenic variants.However, only I2g pathogenic variant was found inhomozygous state and all other mutations wereclearly absent (Figure 4). To know whether thismutation was in homozygous or hemizygous form,MLPA was carried out for detection of deletion.Half dosage was seen in the probes covering exon3,4,6 and 7 indicating heterozygous deletion from

exon 3 to 7. I2g (intron 2 splice) mutation wasfound in homozygous state by the two probesincluded in the MLPA kit P050-C1 for detection ofI2g mutation. (Figure 5).

Her parents were analysed for segregationof mutations by Sanger sequencing and MLPA.Mother was found to carry the I2g mutation asexpected but father was negative for the same. Hewas then checked for deletion by MLPA thatshowed normal dosage for all probes indicatingthat he was negative for the deletion which wasunexpected.

Paternal aunt (sister of proband’s father) waschecked for deletion and duplication by MLPA. Shewas found to harbor a heterozygous duplicationshown by 3 copies of CYP21A2 (Figure 6). Afteranalysing results of paternal aunt, MLPA results ofthe father were reinterpreted and it was inferredthat father harboured both a duplication and adeletion together, due to which he was showingnormal dosage. And his sister had inherited theduplicated allele but not the deletion, and hencewas not a carrier of CAH. Her husband too waschecked and he was found to be negative fordeletion and duplication.

Hence it was confirmed that the proband wascompound heterozygous for whole gene deletionand I2g mutation. The deletion was inherited fromthe father and the I2g mutation from the mother.Proband’s aunt and uncle were counseled aboutthe insignificant risk of having a child affected withCAH.

Patient 2: A five-years-old female child clinicallyconfirmed to have CAH was referred to our geneticclinic for molecular analysis. Her mother was 18weeks pregnant and the family wanted PND to bedone.

Deletions being more common in the CYP21A2gene, MLPA was first done that showedhalf dosage of exon 4,6, and 7 indicatingheterozygous deletion from exon 4-7 (Figure


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Figure 5 MLPA analysis using Coffalyser software showing heterozygous deletion of exon 3- 7 ofCYP21A2 gene in Patient 1. Deletions of exons 3 and 7 are marked by red circle. SALSA MLPA kitP050-C1 was used to detect deletion in our patients. Normal alleles A and C at I2g showingzero value (shown by arrows) indicate absence of both A and C alleles and presence ofhomozygous allele G. Normalized peak height ratio between 0.7 and 1.3 was considered asnormal in patient DNA w.r.t. control DNA.

Figure 6 MLPA analysis using Coffalyser software showing heterozygous duplication indicated by thered circle. All probes fall above the normal ratio (1.5) indicating three copies of CYP21A2 gene inthe paternal aunt of patient 1. SALSA MLPA kit P050-C1 was used to detect deletion in ourpatients. Normalized peak height ratio between 0.7 and 1.3 was considered as normal inpatient DNA w.r.t. control DNA.


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Figure 7 MLPA analysis using Coffalyser software showing half ratios of exon 4-7 indicatingheterozygous deletion from exon 4-7 of CYP21A2 gene in Patient 2. SALSA MLPA kit P050-C1was used to detect deletion in our patients. Normalized peak height ratio between 0.7 and 1.3was considered as normal in patient DNA w.r.t. control DNA.

7). To look for second mutation, Sangersequencing was done and the proband wasfound to harbour c.515T>A (p.Ile172Asn) inexon 4, E6 cluster [c.710T>A (p.Ile236Asn);c.713T>A (p.Val237Glu); c.719T>A (p.Met239Lys)] inexon 6, c.923_924insT (p.Leu306+T) in exon 7,and c.955C>T (p.Gln319Ter) in exon 8, all inheterozygous form (Figure 8).

Her parents were then checked for segregationanalysis to confirm whether these mutations werepresent in cis or trans. Mother was found to haveE6 cluster [p.Ile236Asn, p.Val237Glu, p.Met239Lys],p.Leu306+T and p.Glu319Ter mutations, andfather was heterozygous for the p.Ile172Asnmutation. Hence it was confirmed that the childwas compound heterozygous for the pointmutations.

Discussion

Molecular genetic diagnosis of CAH is morecomplicated than for many other monogenicdisorders due to the location of the CYP21A2gene in the highly variable genomic region withmore than one RCCX repeat unit on thesame chromosome. Presence of a non-functionalpseudogene further complicates the amplification

of the functional gene. The 11 most commonmutations known to cause CAH are present in thepseudogene too. Due to this reason, it is extremelyimportant that the functional gene should only beamplified in the background of pseudogene. It isquite difficult as there is not much differencein the sequence between the two genes. Themost significant difference is the 8 base pairsGAGACTAC present in exon 3 of CYP21A2 and these8 base pairs are deleted in exon 3 of CYP21A1P.This ‘8bp site’ has been exploited extensively todesign primers for selective amplification of theactive gene. To be twice as sure, two primers -forward as well as reverse, were designed at thewild type sequence of the “8bp site” to amplify theCYP21A2 gene into two large fragments. Thisensures that amplification occurs only when bothprimers bind on the wild type sequence at the‘8bp site’. Absence of amplification indicates theabsence of the active gene or presence of thehomozygous 8 bp deletion or presence of only thepseudogene (Figure 3A). The extent of deletion canthen be analysed by MLPA.

It is important to know that due to presence ofthe pseudogene, the capture-based NGS approachis not considered appropriate as it may interferewith the analysis and give erroneous results.Recently, a customized work flow involving


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Figure 8 Partial electropherograms showing mutations detected in Patient 2. A. Mutation c.515T>A inexon 4; B. E6 cluster mutation (c.710T>A, 713T>A, 719T>A) in exon 6; C. c.923_924 insT in exon7; and D. c.955C>T in exon 8 of CYP21A2 gene. All mutations are shown by arrows.

selective amplification of CYP21A2 followed by NGShas been used to correctly detect variants in CAHpatients (Gangodkar et al., 2020).

In Patient 1, all pathogenic variants except I2gwere reported in heterozygous form by NGS.These pathogenic variants appeared in IGV asheterozygous state as half reads were generatedfrom the active gene and half reads from thepseudogene that harboured the correspondingmutant allele. I2g variant was seen in homozygousform as there was no wild type allele presentin the proband. MLPA Kit P050-C1 probes arecomplimentary to the sequences encompassingthe pathogenic variants present in different exons,thus their ratios indicate deletions as well aszygosity of the variants present in the sample. Inthis patient, half ratios of the probes were wronglyinterpreted as heterozygous variants. Since MLPAresults were concordant with NGS results, allvariants were reported without validating bySanger sequencing. However, these ratios wereactually indicating deletions in exons 1-7. Sangervalidation in this patient could have avoided theerroneous interpretation.

The scenario for Patient 2 was completelyopposite to that of Patient 1. In Patient 2, MLPAwas first performed and heterozygous deletionof exon 3 to 7 was detected. Only after

performing Sanger sequencing, the probandwas found to harbour 4 pathogenic variants,[p.Ile172Asn, E6 cluster, p.Leu306+T, p.Glu319Ter],all in heterozygous state. Since there is noprobe available for exon 8 in the MLPA KitP050-C1 used, p.Glu319Ter a common pathogenicvariant present in exon 8 was not picked upby MLPA. Therefore, one should keep in mindwhile analysing the MLPA results that half ratio(0.5) or zero ratio observed in any exonindicates heterozygous or homozygous deletion ofthe corresponding exon respectively. However,these ratios could also indicate the presence ofheterozygous/ homozygous variant in that exon asseen in Patient 2. Thus, MLPA results shouldalways be complemented with Sanger sequencing.On the contrary, whenever homozygous variantsare detected by Sanger sequencing, MLPA shouldbe done to verify whether the pathogenic variantis homozygous or hemizygous.

Therefore, for molecular analysis of theCYP21A2 gene, more than one method should beused for comprehensive analysis. For example,while performing PND for the I2g variant,microsatellite linkage analysis should also beperformed in addition to direct DNA sequencingand MLPA, as this variant is known to have a highrate of allele drop out (Tsai & Lee, 2012)


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Secondly, mutant alleles must be segregated inthe parents to verify their presence on differentalleles for correct interpretation of the moleculargenetics results, as observed in Patient 2. InPatient 1, we could have missed carrier status ofthe father if we had not analysed the proband’saunt. Duplications and deletions of the CYP21A2gene are now being detected relatively frequentlydue to the use of MLPA, a valid alternative toSouthern blotting. However, the interpretationof MLPA results requires extensive knowledgeof CYP21A2 gene rearrangements (Concolino etal., 2009). Duplications have been reported tobe quite frequent in Caucasians (Parajes et al.,2008), however, no data is available from Indiansubjects. Duplications have great impact on thecarrier status of an individual therefore theyrepresent a significant pitfall in the moleculardiagnosis of steroid 21-hydroxylase deficiency(Koppens et al., 2002). Hence, it is imperative toscreen duplications in all couples referred forpreconceptional carrier screening.

References

1. Chen W, et al. Junction site analysis of chimericCYP21A1P/CYP21A2 genes in 21-hydroxylasedeficiency. Clin Chem. 2012; 58: 421–430.

2. Concolino P, et al. Multiplexligation-dependent probe amplification (MLPA)assay for the detection of CYP21A2 genedeletions/duplications in congenital adrenalhyperplasia: first technical report. Clin ChimActa. 2009; 402: 164–170.

3. Concolino P, Costella A. Congenital AdrenalHyperplasia (CAH) due to 21-HydroxylaseDeficiency: A Comprehensive Focus on 233Pathogenic Variants of CYP21A2 Gene. MolDiagn Ther. 2018; 22: 261–280.

4. Dubey S, et al. Prenatal diagnosis of steroid21-hydroxylase-deficient congenital adrenalhyperplasia: Experience from a tertiary carecentre in India. Indian J Med Res. 2017; 145:194–202.

5. Gangodkar P, et al. Clinical application of anovel next generation sequencing assay forCYP21A2 gene in 310 cases of 21-hydroxylasecongenital adrenal hyperplasia from India.Endocrine. 2021; 71: 189–198.

6. Higashi Y, et al. Complete nucleotidesequence of two steroid 21-hydroxylase genes

tandemly arranged in human chromosome: apseudogene and a genuine gene. Proc NatlAcad Sci U.S.A. 1986; 83: 2841–2845.

7. ICMR Task Force on Inherited MetabolicDisorders. Newborn screening for congenitalhypothyroidism and congenital adrenalhyperplasia. Indian J Pediatr. 2018; 85: 935–40.

8. Koppens PF, et al. Duplication of the CYP21A2gene complicates mutation analysis of steroid21-hydroxylase deficiency: characteristics ofthree unusual haplotypes. Hum Genet. 2002;111: 405–410.

9. Narasimhan ML, Khattab A. Geneticsof congenital adrenal hyperplasia andgenotype-phenotype correlation. Fertil Steril.2019; 111: 24–29.

10. Parajes S, et al. High frequency of copynumber variations and sequence variants atCYP21A2 locus: Implication for the geneticdiagnosis of 21-hydroxylase deficiency. PLoSOne. 2008; 3: e2138.

11. Speiser PW, et al. High frequency ofnonclassical steroid 21-hydroxylase deficiency.Am J Hum Genet. 1985; 37: 650–667.

12. Stikkelbroeck NM, et al. CYP21 gene mutationanalysis in 198 patients with 21-hydroxylasedeficiency in The Netherlands: six novelmutations and a specific cluster of fourmutations. J Clin Endocrinol Metab. 2003; 8:3852–3859.

13. Sweeten TL, et al. C4B null alleles are notassociated with genetic polymorphisms in theadjacent gene CYP21A2 in autism. BMC MedGenet. 2008; 9: 1. doi: 10.1186/1471-2350-9-1

14. Therrell B. Newborn screening for congenitaladrenal hyperplasia. Endocrinol Metab ClinNorth Am. 2001; 30: 15–30.

15. Tsai LP, Lee HH. Analysis of CYP21A1P and theduplicated CYP21A2 genes. Gene 2012; 506:261–262.

16. White PC, Speiser PW. Congenital adrenalhyperplasia due to 21-hydroxylase deficiency.Endocrine Rev. 2000; 2: 245–91.

17. Yang Z, et al. Modular variations of thehuman major histocompatibility complexclass III genes for serine/threonine kinaseRP, complement component C4, steroid21-hydroxylase CYP21, and tenascin TNX(the RCCX module). A mechanism for genedeletions and disease associations. J BiolChem. 1999; 274: 12147–12156.


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Genetic Counseling of Prenatally Detected Sex ChromosomeAnomalies

Haseena Sait, Shubha R PhadkeDepartment of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India, 226014

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

Abstract:

Prenatal screening tests are being universallyemployed in the current era to identify womenat risk of fetal aneuploidies. This unveilsa high proportion of unanticipated findingsamongst which sex chromosome abnormalitiesare frequently encountered. It is imperative thatgeneticists and fetal medicine specialists havesufficient knowledge about these anomalies inorder to provide appropriate genetic counselingand assist the couples in decision making duringpregnancy. In the article, we briefly discuss theoutcomes and counseling approach for theprenatally detected common sex chromosomeabnormalities.

Keywords: Sex chromosome anomalies, prenatalscreening,Turner syndrome, Klinefelter syndrome,genetic counseling.

Introduction

Sex chromosome abnormalities (SCAs) arethe most frequently encountered chromosomalabnormalities both prenatally and at birth. Theseare due to the presence of an extra or missing X orY chromosome and most commonly include 45,X;47,XXX; 47,XXY; and 47,XYY. The prevalence ofSCAs is estimated to be around one in 500newborns, twice as common at birth as trisomy21. The frequency at prenatal diagnosis is muchgreater and ranges from 1 in 250 to 300 (Lindenet al., 2002). Though not a primary target fordetection in prenatal diagnosis, incidental findingslike SCAs cannot be avoided. With expanding useof population wide screening for chromosomalanomalies by novel genomic technologies likenon-invasive prenatal screening (NIPS), suchproblems will be more commonly seen in the nearfuture. Being an unexpected finding in prenataltesting, SCAs pose significant challenge to the

genetic counselor in terms of counseling anddilemma for the family. The outcome varies greatlyfrom normal phenotype to those with significantphenotypic abnormalities. Individuals with SCAusually do not have significant intellectualdisability. Hypogonadism and infertility remain themajor issues; both of which have solutions inthe form of hormone replacement therapy (HRT)and assisted reproductive techniques (ART). Thedifficulties in decision making are obvious asuncertainties about the phenotype are not strongenough to consider termination of pregnancy.The decision depends upon parents’ familyhistory and their perspectives to look at theproblem. The parents’ thinking gets influencedby what is conveyed to them by health careprofessionals involved in prenatal diagnosis andcounseling. Hence, it is essential that accurate andup-to-date information about the likely outcomesis communicated to the family in a simplifiedmanner. Through this article, we describe theoutcomes of various prenatally detected SCAs andthe issues in counseling for the same.

The following case scenarios present somecommon problems faced by the clinicians andfamilies and perspectives in approaching them:

Case scenario 1: A 32-year-old G2P1+0L1 motherwho has a previous child with Down syndrome(Trisomy 21) visits us at 16 weeks of gestationfor prenatal counseling. After pre-test counselingregarding the risk of recurrence of 1% for trisomy 21in the current pregnancy, she opts for prenataltesting. Amniocentesis followed by quantitativefluorescent polymerase chain reaction (QFPCR)reveals 47,XXY and this finding is confirmed bykaryotyping.

Case scenario 2: A 35-year-old G3P0+2, withprevious two abortions, presents with history of twoIVF (in vitro fertilisation) failures and consults us inview of non-invasive prenatal screening (NIPS) testshowing high risk for monosomy X. This finding is


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confirmed by chromosomal analysis from amnioticfluid. Ultrasonography evaluation at 18 weeks isnormal.

Case scenario 3: A 30-year-old G3P2 mother withprevious two healthy children, visits us at 17 weeksfor counseling regarding high risk for trisomy 21(1:151) on quadruple marker testing. Pre-testcounseling is provided. Options of NIPS and invasivetesting are given and she opts for invasive testing.Amniocentesis followed by QFPCR and karyotype issuggestive of 47,XXX chromosome complement.

Counseling for the above-mentioned casesrequires in-depth knowledge about the clinicalphenotypes of SCAs, variability in presentationand availability of management options forhypogonadism and infertility. As these situationsare not infrequent, it is important thatclinical geneticists, fetal medicine specialists andcounselors acquire adequate knowledge toprovide prospective parents with sufficient andunbiased information regarding these SCAs andguide them in decision making.

Pre-test Counseling

Pre-test counseling for prenatal proceduresdone for varied indications should alwaysinclude discussion about the various disorderswhich can be detected by the test. A briefdiscussion on the outcome of these disordersin general which would result in mentalor physical abnormalities should be discussed.The counseling must include the possibility ofdetection of unrelated abnormalities includingSCAs, unbalanced autosomal abnormalities otherthan the intended ones, and mosaic forms. Manyof these may have variable outcomes. Somegroups even suggest that obtaining consent fromcouples, as to whether to include or exclude theresults of these incidental findings, is essential(Herlihy et al., 2010).

Post-test counseling

Post-test counseling should mainly focus on thespecific disorder which has been diagnosed. Thefollowing points have to be kept in mind whenproviding information and counseling to thecouples:• couple should be made aware of the

frequency of the condition in the generalpopulation;

• the occurrence of SCAs is a random event;• incidentally detected sex chromosomeaneuploidies are more often associated withnormal to mildly affected phenotypes thanpostnatally detected SCAs (Pieters et al.,2011);• the possibility of spontaneous abortion ofpregnancy especially in fetuses with 45,Xshould also be mentioned;• variability in the phenotype of the conditioncan exist and the inability to provide a preciseindividual prognosis must be discussed;• uncertainty and complexity in providingcounseling in case of mosaicism for SCAsshould be discussed;• role of other autosomal genes andenvironmental factors altering a child’sprognosis should be stressed upon;• written material providing comprehensiveinformation about the relevant karyotype willbe useful;• if possible, showing selected photographs ofindividuals with SCAs and talking to otherparents of children with SCAs can bereassuring and helpful;• finally, the issue of disclosure of SCAdiagnosis by parents to others and theconsequences of the same should beaddressed at the time of diagnosis;• if the couple decides to continue withthe pregnancy, they should be adequatelycounseled regarding when and how toanticipate the problems and to seek medicalcare;• the couple should be informed ofthe available postnatal interventions. Thepotential benefit of knowledge of thecondition to facilitate early interventionshould be highlighted; and• it is important to be aware that in addition tophenotypic outcome, the obstetric history ofthe woman will play an important role intaking decision about the fate of the currentpregnancy. This can be perceived in the casescenarios discussed above.

Apart from the above-mentioned points forgeneral counseling, the major point that hasto be highlighted in the discussion of theseSCAs should be its impact on the reproductive


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Table 1 The prenatal and postnatal outcome of few of the commonly detected sex chromosomeanomalies.

45,X(Morgan, 2007)

47,XXY(Girardin & Van

Vliet, 2011)

47,XXX(Wigby et al.,

2016)

47,XYY(Bardsley et al.,

2013)Prevalence 1:2500-3000 live born

girls1:500-1000 live bornmales

1:1000 live bornfemales

1:1000 live bornmales

Risk factor None Advanced maternalage

None None

Prenataloutcome

99% get abortedspontaneously;increased nuchaltranslucency (NT),cystic hygroma orhydrops

High rates of pretermdeliveries; no specificantenatalmalformations

No specificantenatalmalformations

No specificantenatalmalformations

Intelligence Normal but 15-20points below controlsand siblings

Normal but 15-20points below controlsand siblings

Normal but 15-20points belowcontrols andsiblings

Normal

Characteristicfeatures

Short stature (>95%),webbed neck, lowposterior hairline,narrow palate withcrowded teeth, broadchest with widelyspaced nipples,cubitus valgus,multiple pigmentednevi

Tall stature, smalltestes, gynecomastiain late puberty,sparse body hair

Tall stature Tall stature,macrocephaly,macrodontia,scoliosis

Associatedabnormalities

Cardiacmalformation(coarctation of aortaor bicuspid aorticvalve in 75%)sensorineuralhearing loss,recurrent otitismedia, renalmalformation (e.g.,horseshoe kidney,duplicated or cleftrenal pelvis),autoimmunethyroiditis, celiacdisease, scoliosis

Diabetes, metabolicsyndrome,osteoporosis andcardiovasculardiseases inadulthood

Rare Hand tremors orotherinvoluntarymovements(motor tics),seizures, andasthma

Development At risk of mild delayin acquiringnonverbal, social, andpsychomotor skills

Reduction in speech,language abilities,verbal processingspeed and schoolperformance

Mild motor delay,languagedifficulties anddecreased schoolperformance

At risk for mildspeech/languageand motordelays, learningdisabilities


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Psychiatricailment#

Prone for shyness,anxiety, lowself-esteem anddepression

Depression,paraphilia, autisticand obsessive-compulsive arecommon

Psychotic illnesslike cyclothymicand labilepersonalitydisorder common(38%)

Attention deficit(50%), autismspectrumdisorder (29%)and anxiety(26%)

Puberty Absent* Normal(hypogonadismoccurs later)

Normal Normal

Reproduction Infertile* Infertile (options likesperm extraction andcryo-conservationavailable)

Fertile (4%developprematureovarian failure)

Normal

Management& follow up

Echocardiogram &renal ultrasound atbirth; annualphysical,psychological,cardiac, thyroid, boneand blood pressureevaluation; hormonaltherapy atadolescence

Annual physical andpsychologicalevaluation;endocrinologicalevaluation atadolescence;testosterone therapyduring adolescence

Annual physical &neuropsychologi-cal evaluation;ovarian functionassessmentduring earlyadulthood

Annual physical& neuropsycho-logicalevaluation

Risk ofrecurrence

Rare Rare Rare Rare

*Normal menstruation and fertility seen in 2-5% mosaic individuals# May be seen more frequently than in the general population

and neurocognitive outcomes. These issues arediscussed briefly in Table 1. Though not alwaysfoolproof, the following issues can be discussed inbrief in selected scenarios.

1. 45,X (Turner syndrome):• Mental development and cognition areusually normal.• Major concern for this conditionis hypogonadism and primaryamenorrhea. Hormone replacementtherapy (HRT) is indicated to initiate andmaintain secondary sexual characters.• Short stature is common. Early growthhormone therapy can help to improveshort stature.• Associated abnormalities in cardiovas-cular system and renal system shouldbe mentioned. Some of the cardiacanomalies can be detected by prenatalechocardiography but coarctation ofaorta, commonly seen in girls with

Turner syndrome is difficult to diagnoseprenatally.• Risk of infertility is high. With the helpof assisted reproductive techniques,pregnancy can be achieved in somewomen with Turner syndrome.

2. 47, XXY (Klinefelter syndrome):• These individuals may have mildcognitive and psychiatric disturbances.• Major issue is male hypogonadism.Treatment with sex hormones forhypogonadism is indicated.• Infertility is common but reproductiveoptions like testicular sperm extraction(TESE) and cryo-conservation arepossible to improve reproductiveoutcomes.

3. 47,XXX and 47,XYY:• The reproductive and cognitive outcomeis usually satisfactory.


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• The psychological and behaviouralproblems reported in studies are usuallymild and these individuals have a slightlyincreased prevalence when comparedto normal individuals but ascertainmentbias behind these studies should bekept in mind.

Previously, phenotypes of SCAs were knownonly for postnatally detected cases as they arethe only ones who seek medical attention forphenotypic abnormalities. This ascertainment biasreflected in counseling for SCAs where incidentallydetected SCAs during prenatal tests led totermination of most of these pregnancies. Thisissue was compounded by lack of adequateinformation about long term follow up of childrenwith SCAs who were diagnosed prenatally.However, eliminating such ascertainment bias,recent studies have proved that incidentallydetected prenatal diagnosis of SCAs is associatedwith normal to mildly affected phenotypes whencompared to postnatal cases (Pieters et al., 2011).

Studies have evaluated parental attitudetowards terminating or continuing a SCA-affectedpregnancy and have found that factors like specifictype of SCA, parental age, gestational week atdiagnosis, counselor’s genetic expertise, numberof children in the family, previous experience ofthe family with children having birth defectsor genetic disorders, socioeconomic status, andethnicity and religious beliefs, influenced thedecision to continue or abort the pregnancy.History of infertility or previous child withdevelopmental delay may also complicate thedecision-making process. This also gets largelyinfluenced by the information one receives from ahealth professional (Operto et al., 2019; Shaw etal., 2008; Jeon et al., 2012). In recent times, therehas been an emerging trend towards continuationof pregnancy of a fetus with SCAs due toimproved counseling efforts and availability ofadequate information on prognosis of these SCAs.Simultaneous progress in the field of ART has alsototally changed the reproductive outcome of theseindividuals with SCAs.

Genetic counseling for sexchromosomal mosaicism

Mosaicism is defined as the presence of two ormore cell lines derived from a single zygote butwith different chromosomal complements in an

individual. Genetic counseling becomes complexin such cases due to variability in phenotypicexpression due to variable degree of mosaicism indifferent tissues. These factors pose uncertaintyabout the postnatal outcome of such disorders.

In prenatally detected 45, X/46, XY mosaicism, anormal male phenotype was present in 90% ofcases (Telvi et al., 1999). However, the dilemmain counseling exists as in 10% of cases, thephenotypic spectrum can vary from femaleswith Turner syndrome to males with infertilityor individuals with ambiguous genitalia. Theneurodevelopmental and reproductive outcomewill also be highly variable in these individualsposing significant challenges in counseling.

A favourable prognosis exists for mosaicTurner syndrome (45,X/46,XX) who tend to havefewer signs and health problems like near normalstature and may have normal reproductivecapabilities and no cardiovascular complications(Tuke et al., 2019). Similarly, mosaic Klinefeltersyndrome are well androgenized and have betterreproductive capability than their non-mosaiccounterparts (Samplaski et al., 2013).

Genetic counseling for structuralaberration of sex chromosomes

Structural aberrations involving X chromosomecommonly include isochromosome Xq and ringchromosome. For such structural aberrationsinvolving one X chromosome, the counseling issimilar to that for Turner syndrome. However, ringX chromosome may be associated with moresevere intellectual disability.

Cytogenetically visible structural aberrations ofY chromosome include deletions, translocations,rings, inversions and isochromosomes. Structuralaberrations of Y chromosome usually result inmosaicism due to its predisposition to subsequentchromosome instability and loss of the abnormal Ychromosome, thereby causing mosaic 45,X. Thephenotypes in such case can vary from femaleswith Turner syndrome to males with infertility orambiguous genitalia based on number of cellslines with 45,X and abnormal Y chromosome(Patsalis et al., 2005). Counseling in these cases ischallenging as a definite prediction of phenotypeis impossible and this uncertainty is likely to causedilemma in decision-making for the family.

Not all structural aberrations are pathogenic.Pericentric inversions involving Y chromosomeare mostly familial and not associated with any


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phenotypic manifestations or fertility issues exceptin rare cases when genes determining sex in theinverted area are disrupted (Motos Guirao, 1989).

For other rare and complex aberrations, acomprehensive use of cytogenetic, microarrayand fluorescent in situ hybridisation techniquesare required for accurate identification of suchabnormalities. The counseling for these rare SCAsvaries on a case-to-case basis and is beyond thescope of this article.

Other rare SCAs

Chromosomal abnormalities where there ispresence of more than two X chromosomes-

48,XXY or 49,XXXXY: They are more severelyaffected in terms of neurocognitive andbehavioural function. The phenotype progressivelydeviates from normal as the number of Xchromosome increases. These individuals havebeen shown to function at a lower cognitivelevel and with more immature and maladaptivebehaviours as compared to individuals with fewerX chromosomes (Visootsak et al., 2007). Infertilityand inadequate virilization are anticipated.

Conclusion

The chances of encountering SCAs are high withwidespread availability of prenatal tests andespecially after widespread use of NIPS in obstetricpractice. It is therefore crucial that geneticists andcounselors acquire adequate knowledge regardingthe implications of SCAs and develop structuredpre-test and post-test counseling strategies. Thisin turn would help prospective parents to take apersonalized and autonomous decision regardingthe pregnancy.

References

1. Bardsley MZ, et al. 47,XYY syndrome: clinicalphenotype and timing of ascertainment. JPediatr. 2013; 163: 1085–94.

2. Girardin CM, Van Vliet G. Counseling of acouple faced with a prenatal diagnosis ofKlinefelter syndrome. Acta Paediatr. 2011; 100:917–922.

3. Herlihy AS, et al. Assessing the risks andbenefits of diagnosing genetic conditionswith variable phenotype through population

screening: Klinefelter syndrome as anexample. J Community Genet. 2010; 1: 41–46.

4. Jeon KC, et al. Decision to abort aftera prenatal diagnosis of sex chromosomeabnormalities: A systematic review of theliterature. Genet Med. 2012; 14: 27–38.

5. Linden MG, et al. Genetic Counseling for SexChromosome Abnormalities. Am J Med Genet.2002; 110: 3–10.

6. Morgan T. Turner syndrome: diagnosis andmanagement. Am Fam Physician. 2007; 76:405–410.

7. Motos Guirao MA. Pericentric inversion of thehuman Y chromosome. An Esp Pediatr. 1989;316: 583–587.

8. Operto FF, et al. Cognitive profile,emotional-behavioral features, and parentalstress in boys with 47,XYY syndrome. CognBehav Neurol. 2019; 32: 87–94.

9. Patsalis PC, et al: Identification of highfrequency of Y chromosome deletions inpatients with sex chromosome mosaicism andcorrelation with the clinical phenotype andY-chromosome instability. Am J Med Genet A.2005; 135: 145–149.

10. Pieters JJ, et al. Incidental prenatal diagnosisof sex chromosome aneuploidies: health,behavior, and fertility. ISRN Obstet Gynecol.2011; 2011: 807106.

11. Samplaski MK, et al. Phenotypic differences inmosaic Klinefelter patients as compared withnon-mosaic Klinefelter patients. Fertil Steril.2014; 101: 950–955.

12. Shaw SW, et al. Parental decisions regardingprenatally detected fetal sex chromosomalabnormality and the impact of geneticcounseling: An analysis of 57 cases in Taiwan.Aust N Z J Obstet Gynaecol. 2008; 48:155–159.

13. Telvi L, et al. 45,X/46,XY mosaicism: Report of27 cases. Pediatrics. 1999; 104: 304–308.

14. Tuke MA, et al. Mosaic Turner syndromeshows reduced penetrance in an adultpopulation study. Genet Med. 2019; 21:877–886.

15. Visootsak J, et al. Behavioral phenotype of sexchromosome aneuploidies: 48,XXYY, 48,XXXY,and 49,XXXXY. Am J Med Genet Part A. 2007;143A: 1198–1203.

16. Wigby K, et al. Expanding the Phenotype ofTriple X Syndrome: A Comparison of PrenatalVersus Postnatal Diagnosis. Am J Med Genet A.2016;170: 2870–2881.


GeNeXprESS

Methylation, Monogenic Disorders and MoreVarun Venkatraghavan MS

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

Correspondence to: Dr Varun Venkatraghavan MS Email: [email protected]

Determining gestational age usinggenome methylation profile: A novelapproach for fetal medicine (Falick Michaeli

et al., 2019)

The need for accurate gestational age of aneonate need not be stressed. The availablemethods have limitations in various settings. Inthis study, the methylation status was studiedusing reduced representation bisulfite sequencing(RRBS) in DNA extracted from cord blood andplacenta. The investigators identified a set of332 differentially methylated regions (DMRs) thatundergo demethylation in late gestational ageand a set of 411 DMRs that undergo de novomethylation in late gestational age. The data ofsamples used for training (5 less than 33 weeksand 5 more than 33 weeks) was used to evaluate41 ‘test’ samples of neonates from 25 to 40 weeks.A neonatologist using Ballard criteria, assessedthe gestational age of the neonates. This studyshows that this novel method (RRBS) of studyingmethylation levels in DNA of white blood cells incord blood provides an accurate assessment ofgestational age and can be used in clinical settings.It seems the design of the epigenetic clock isworking and is useful.

DNA methylation signature for EZH2functionally classifies sequencevariants in three PRC2 complex genes(Choufani et al., 2020)

Weaver syndrome belongs to the groupof overgrowth/intellectual disability syndromes(OGID), and is caused by mutations inEZH2 gene. EZH2 codes for a part of the

catalytic component of the polycomb repressivecomplex 2 (PRC2) that regulates genome-widechromatin and gene expression by methylationof lysine 27 of histone H3. EED and SUZ12which cause Cohen-Gibson syndrome andImagawa-Matsumoto syndrome respectively, areother components of PRC2. This study usedgenome-wide DNA methylation (DNAm) data for187 cases with OGID and 969 control subjects,and demonstrated that pathogenic variants inEZH2 produce highly specific and sensitive DNAmsignature reflecting the phenotype of WS. Thesignature identified differentiates loss-of-functionfrom gain-of-function missense variants andto detect somatic mosaicism. Loss-of-functionleads to decreased methylation of promotorregion thus causing overgrowth phenotypeand gain-of-function mutations cause growthrestriction due to increase in methylation. Thissignature identified in EZH2 helps classifysequence variants in EED and SUZ12 as well, andcan predict presence of pathogenic variants inundiagnosed individuals with OGID.

DNA methylation at birth predictsintellectual functioning and autismfeatures in children with Fragile Xsyndrome (Kraan et al., 2020)

Fragile X syndrome (FXS) has some uncommoncharacteristics of molecular pathology includingdynamic mutation of increase in the number oftriplet repeats in the 5´ untranslated region, andsilencing of the gene by epigenetic modificationsof the FMR1 promoter including DNA methylation(DNAm). Some other characteristics seen in FragileX syndrome, that are unusual for a monogenicdisorder, are premutation, tissue to tissuevariation in number of repeats and methylation


GeNeXprESS

leading to mosaicism including premutation/mutation mosaicism. The level of DNAm of FragileX Related Epigenetic Element 2 (FREE2) locatedto exon1/intron1 of FMR1 gene has been seento correlate with FMR protein and intellectualfunction. This study assessed DNAm of FREE2by Methylation Specific-Quantitative Melt Analysis(MS QMA) and the EpiTYPER system, in storednewborn blood spots (NBS) and newly createddried blood spots (DBS) from 65 children with FXS.Good correlation was shown between DNAm andneurocognitive function including autism; more soin males than in females. Correlation was alsoobserved between DNAm and FMR mRNA. Inmales with FXS the change in the level of FREE2mfrom birth to childhood was not significant butdecrease in the level of FREE2m was observed inFXS females. The stochastic changes may be thecombined effects of environmental factors, clonalselection and mRNA toxicity. The results havepotential for using FREE2M for newborn screeningas well as for more accurate prognostication.

MeCP2 links heterochromatincondensates and neurodevelopmentaldisease (Li et al., 2020)

Rett syndrome is a neuro-developmental disorderseen in females which is caused by mutations inMECP2 gene associated with DNA methylationregulation. MeCP2 binds methylated DNA,thus regulating transcription and chromatinorganization. As per the concept called phaseseparation, certain molecules form large dropletsby which the molecules inside the droplet areseparated from the rest of the cell; these dropletsare called condensates. By using fluorescentlabelled MeCP2 and HP1 proteins, live cell imagingwas done which showed that the two proteinsoccur in the same heterochromatin condensates.

Imaging of neurons of mice expressing GFP-taggedMeCP2 protein from the endogenous locusrevealed that MeCP2-GFP occurs in Hoechst-denseheterochromatin. These results indicate thatMeCP2 is a dynamic component of liquid-likeheterochromatin condensates in murine braincells. The scientists have done variousexperiments to understand physicochemicalproperties of MeCP2 and effects of mutationson the heterochromatin condensates. Basedon the results the authors propose that alarge number of MeCP2 molecules, usingmultiple weak and dynamic interactions, formmembrane-less bodies that can concentrateand compartmentalize additional componentsengaged in heterochromatin function. Rettsyndrome mutations cause decrease in theprotein as well as alter the condensate properties.The understanding of molecular pathology mayhelp in development of new approachesto pharmacological modification of condensatebehaviors for the treatment of Rett syndrome.

References

1. Choufani S, et al. DNA Methylation Signature forEZH2 Functionally Classifies Sequence Variantsin Three PRC2 Complex Genes. Am J HumGenet. 2020; 106: 596–610.

2. Falick Michaeli T, et al. Determining gestationalage using genome methylation profile: A novelapproach for fetal medicine. Prenat Diagn.2019; 39:1005–1010.

3. Kraan CM, et al. DNA Methylation at BirthPredicts Intellectual Functioning and AutismFeatures in Children with Fragile X Syndrome.Int J Mol Sci. 2020; 21: 7735.

4. Li CH, et al. MeCP2 links heterochromatincondensates and neurodevelopmental disease.Nature. 2020; 586: 440–444.

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Volume 14 | Issue 3 | July - September 2021 Genetic