ESSENTIA

      

AL GENETTICS FOR O 

  

OBSTETRICCIANS

Review Article

BACKGROUND

Genetic diseases are not as rare as once believed. In fact,genetic disease is a major cause of illness and death.Approximately 2% to 3% of all pregnancies result in aneonate with a serious genetic disease or a birth defect thatcan cause disabilities, mental retardation, and in some casesearly death. Genetic factors are present from conception,and their expression may vary throughout development,whereas environmental influences are changing constantly.Many conditions previously thought to be nongenetic arenow understood to be multifactorial diseases with thecontribution of various genetic and environmentaldeterminants being recognized increasingly.

A genetic disorder is a disease that is caused by anabnormality in an individual’s DNA. Abnormalities canrange from a small mutation in a single gene to the additionor subtraction of an entire chromosome or set ofchromosomes. Genetically determined disorders often aresubdivided into 3 major groups: single gene, chromosome,and multifactorial (polygenic) diseases. Somatic cellgenetic defects play a role in human cancer and constitute afourth group.

Single-gene disorders

This type is caused by changes or mutations that occurin the DNA sequence of one gene. Individually single genedisorders are rare (1 in 10,000-15,000) but collectively theycan affect upto 1-2% of all births. Some conditions are

highly prevalent in selected populations like sickle celldisease in Africans, thalassemia in the geographical beltextending from the Mediterranean countries to South EastAsia. Since the late 1970s, the number of disordersclassified as single gene has increased from an estimated2500 to approximately 14,000 (as of April 2003). Of these14,000 single gene disorders, 93.7% are classified asautosomal, 5.6% as X-linked, and 0.7% as other [1].Considering that the human genome consists ofapproximately 30,000 genes, the number of diseasesclassified as monogenic is expected to increase. Comparedto general population, the risk of occurrence of geneticdiseases in affected families is very high depending on thepattern of inheritance. However, previous occurrence ofthe disease in the family is not necessary. The defect mayarise de novo for the first time in any individual or theremay be silent carriers in the family who give birth to anaffected child without a positive family history (autosomalrecessive disorder). The four most common patterns ofmendelian inheritance are based primarily on two factors:on which type of chromosome (autosome or sexchromosome) the gene locus is found and whether thephenotype is expressed only when both chromosomes of apair carry the abnormal allele (recessive) or whether thephenotype can be expressed when just one chromosomecarries the mutant allele (dominant). Single-gene disordersare inherited in recognizable patterns: autosomaldominant, autosomal recessive, and X-linked.

If a person carries the dominant gene for a disease, he

251 Apollo Medicine, Vol. 6, No. 3, September 2009

ESSENTIAL GENETICS FOR OBSTETRICIANS

Neerja GuptaConsultant Clinical Genetics, Department of Genetics, Indraprastha

Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.e-mail- neerjaagarwal@yahoo.co.in

Clinical genetics is one of the most rapidly advancing fields in medicine. Spectacular progress has beenachieved in this century with unravelling of the entire draft sequence of the human genome. A majorcontribution of these advances has been in diagnosis, management and prenatal diagnosis of geneticdisorders as treatment in most cases is difficult or impossible and where available beyond the means of mostfamilies. Genetic technology is advancing rapidly, bringing new, safer and more sensitive ways to diagnosegenetic conditions pre- and postnatally. These advances will bring about profound changes in the way wedeliver obstetric services to women and their families. Diagnosing a genetic disorder not only allows fordisease-specific management options but also has implications for the affected individual’s entire family.Hence, a working understanding of the underlying concepts of genetic disease is important for all practicingclinicians. Although it is impossible to know all aspects of clinical and molecular genetics, basic knowledge ofcertain topics is a must for all practicing obstetrician/gynecologists.

Key Words: Genomics, Prenatal diagnosis, Genetics, Genetic disorders.

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or she will usually have the disease and each of the person’schildren will have a 1 in 2 (50%) chance of inheriting thegene and getting the disease. Diseases caused by adominant gene include achondroplasia (a form ofdwarfism), Marfan syndrome (a connective tissuedisorder), and Huntington disease (a degenerative diseaseof the nervous system).

People who have one recessive gene for a disease arecalled carriers, and they don’t usually have the disease butthey are at risk of producing children with autosomalrecessive diseases (Fig. 1) such as Cystic fibrosis, Sicklecell anemia , and thalassemia are caused by recessivedisease genes from both parents coming together in a child.

Some recessive genetic variants are carried only on theX chromosome, which means that usually only boys candevelop the disease because they have only one Xchromosome. Girls have two X chromosomes, so theywould need to inherit two copies of the recessive gene to getthe disease. Examples are hemophilia and Duchennemuscular dystrophy.

In X-linked dominant inheritance, both male andfemale children have a 1 in 2 risk of inheriting the mutantallele from the affected mother and thus being affected aswell. Sons of an affected male do not inherit the condition,

whereas all daughters are affected clinically.

Chromosomal

Chromosomes are carriers of genetic material. Anyabnormality in chromosome structure as missing or extracopies or gross breaks and rejoining (translocations) canresult in disease.

Some chromosome anomalies are “balanced” andinclude the full complement of genetic material in arearranged form. Although balanced rearrangements havebeen associated with infertility and medical complications(perhaps because of breakpoints in important genes orsecondary to positional effects of gene expression), mostpeople with balanced chromosome rearrangements arehealthy.

Statistically, numerical chromosome abnormalities arethe most common type of chromosome disorder.Chromosome aneuploidy occurs when there is other than amultiple of the typical haploid set. About 60% of thechromosomal abnormalities are spontaneously aborted inthe first trimester. This prevalence goes downapproximately 5% in the late abortions and stillbirths. Atbirth, only 0.6% of the newborns have been found to have achromosomal abnormality. Although trisomy 16 is the mostcommon autosomal trisomy in miscarriages, trisomies 21(Down syndrome), 18 (Edwards syndrome), and 13 (Patausyndrome) are seen at considerable frequencies innewborns. Of these, Down syndrome or trisomy 21 is thecommonest one. Notably, the risk of having a newborn withany of these chromosome trisomies increases with maternalage, although not all chromosome aneuploidy is associatedwith maternal age. Turner syndrome (45, X) is most oftencaused by loss of the paternal X chromosome and is presentin 1% of all conceptions; however, 98% result inmiscarriage. Chromosome polyploidy occurs when thenumber of chromosome sets is other than two. The mostcommon type of chromosome polyploidy is triploidy (69chromosomes), present in 1% to 3% of all conceptions.Triploidy is a sporadic occurrence and most commonlyhappens when 1 haploid egg is fertilized by 2 haploidsperm.

Another group of chromosome disorders includes thoseresulting in genetic imbalance despite retention of thenormal number of 46 chromosomes. This group includeschromosome translocations in their unbalanced form,deletions, and duplications. In these situations, there issome net loss or gain of genetic material.

Chromosomal disorders are often suspected by thepresence of mental retardation, facial dysmorphism,multiple congenital abnormalities, and failure to thrive.Fig.1. Autosomal recessive inheritance

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Standard chromosome analysis using G-banding allowsonly the detection of relatively large structural rearrange-ments (3-4 megabases) and depends on the band resolution.

Multifactorial

Many genetic disorders appear familial but do notfollow a single gene pattern of inheritance. Thesemultifactorial (polygenic, complex) disorders are the resultof a combination of alterations in multiple genes withvarying degrees of effect that act in concert withenvironmental factors, thus producing a clinical phenotypewhen a developmental threshold is reached [2]. Thesedisorder occur with high frequency in close relatives ascompared to the general population. Examples includeheart disease, hypertension, Alzheimer’s disease, arthritis,diabetes, and obesity. Another important group ofmultifactorial disorders is congenital malformation. Recentadvances in various molecular techniques like array CGHetc have opened the possibility of identifying major genesthat can predispose to these disorders.

Genetic Counseling

An accurate diagnosis of the disorder is very essentialfor any genetic counseling [3-5]. It is defined as “theprocess by which patients or relatives at risk of a disorderare advised of the consequences of the disorder, theprobability of developing and transmitting it, and ways inwhich this can be ameliorated” [1]. It also helps theindividual or family to choose a course of action whichseems to them appropriate in view of their risk, their familygoals, and their ethical and religious standards and act inaccordance with the decision and also to make the bestpossible adjustments to the disorder in an affected familymember and/ or to the risk of recurrence of that disorder.

There are certain situations which can be identifiedbefore or after conception in which genetic counseling andprenatal diagnosis may be required. These indications are

• Advanced maternal age (>35 years)

• Recurrent miscarriages (3 or more) / Infertility /primary ammennorhea

• Previous child with

– dysmorphism /single or multiple malformationslike cardiac renal, brain defects/short stature/neuromuscular disorder/neurogenetic disorder/Metabolic disorder/Unexplained MR/CerebralPalsy / autism / Chromosomal abnormality /Deafness/ thalassemia/Hemophilia

• Previous unexplained still birth/s, neonatal or

infantile deaths with or without congenitalmalformations

• Family history of a genetic disorder like anychromosomal abnormality like Down syndrome,thalassemia, spinal muscular atrophy, hemophilia,congenital deafness or Gaucher disease

• Consanguinity especially with a history of suspectedgenetic disorder

• Maternal disease like diabetes, hypothyroidism toidentify high risk fetuses through level II ultrasound

• Positive maternal serum screen either first or secondtrimester/Abnormal fetal ultrasound

• Exposure to known or suspected teratogen duringpregnancy

• Amniotic fluid abnormalities in second/third trimesterespecially in association with growth retardation

• Maternal Infection (TORCH infection)

Steps in an antenatal case Management [3,4]

The skills required to make a genetic diagnosis aresimilar to those used for more common health problems,including history taking, physical examination, and properlaboratory testing.

History- The pregnancy history of the patient’s mothermight disclose maternal disease potentially causative of orrelated to the fetal condition, as seen in certain metabolicdisorders such as untreated maternal PKU or fatty acidoxidation disorders. Sometimes, maternal disorders(diabetes) environmental or drug exposures (valproate,warfarin etc) during pregnancy can cause multiplemalformations such as in fetal valproate syndrome orwarfarin embryopathy. Medical history of maternaldisorders like SLE, hypothyroidism is also important forbetter fetal outcome.

Family History - A thorough family history includesdetailed information on relatives’ ages, current and pastmedical health (including developmental or learningproblems), birth defects, obvious dysmorphism, andsurgeries. Specifically, questions about miscarriages,stillbirths, and infant deaths, as well as infertility, should beasked. For deceased family members, age and cause ofdeath should be documented. The racial and ethnicbackground is of importance in identifying higher riskgroups. In addition, the possibility of consanguinity in thefamily history should be explored when clinically relevant.Drawing a family tree (pedigree) that symbolically (Fig.2)

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represents the family and demonstrates relationshipsbetween affected family members is an efficient and highlyinformative exercise.

History of any genetic disorders in the family- Familyphotographs or medical records may be of help, particularlyif other family members are suspected to have the samegenetic disorder.

Examination of the couple is required especially whenthere is a family history of a particular genetic disorder likeneurofibromatosis, tuberous sclerosis, or incontinentiapigmenti.

Investigations are done based on the indications apartfrom routine antenatal screening.

Specialized investigation

The importance of precise diagnosis for geneticcounseling cannot be over emphasized. However,specialized tests like chromosomal analysis, enzymeanalysis, and DNA analysis are required to arrive at a finaldiagnosis. Before these tests are ordered, informationshould be obtained on the type and volume of the specimenrequired (blood, urine, fibroblasts, amniocytes), type oftube in which the specimen should be kept, andconditions under which the specimen should be sent(Appendix- A).

DNA based tests (Molecular tests)

DNA testing investigates alterations in a gene that resultin disease. Confirmed molecular diagnosis in index casewould also help in carrying out prenatal diagnosis (byamniocentesis or chorionic villi sampling) for therespective disorder. Unless the type of mutation/s in theproband or carrier parents is identified, prenatal diagnosisis not feasible. It should preferably be identified before nextpregnancy. Examples of widely available moleculargenetic tests include thalassemias , muscular dystrophies,spinal muscle atrophy, Fragile X syndrome, hemophilia Aand B, cystic fibrosis, albinism, achondroplasia etc.

Chromosomal analysis (Cytogenetics)

Chromosomal abnormalities can be diagnosed afterbirth using a blood test, or before birth using prenatal tests(amniocentesis or chorionic villi sampling). Tissues mostcommonly used are lymphocytes and amniocytes. Anyabnormal finding has its own implication and management.Cytogenetic analysis on bone marrow also helps indiagnosis and prognosticating, especially in cancers. Ittakes on average 1 to 3 weeks to obtain a definitive result,the time depending on the method.

Newer diagnostic techniques include: (i) Rapid-FISH(rapid fluorescence in situ hybridization); (ii) MLPA(multiple ligation PCR amplification), and (iii) QF-PCR(Quantitative Fluorescent Polymerase Chain Reaction).

These methods of analysis do not require culturing, theamount of the sample material may be very small and theresult is obtained in just few days. In comparison, classicalcytogenetic analysis (karyotyping) after amniocentesisrequires 15-20 mL of amniotic fluid, culturing of fetal cells(amniocytes) and takes around 10 to 21 days to produce theresult.

Biochemical testing

Biochemical testing refers to analyses of metabolitesthat are either the substrates or the products of a deficientenzyme. Thus, increases or decreases of metaboliteconcentrations are indirect indicators of metabolicdisorders caused by an enzyme deficiency.If abnormalmetabolites are identified, the disease may then beconfirmed by enzyme analysis when available like inmucopolysacharidoses, Gaucher disease, Tay Sachsdisease, etc. Enzyme analysis often requires a fibroblastculture or a fresh liver biopsy. Some enzyme tests can bedone on serum, red blood cells, or leukocytes.

PRENATAL SCREENING AND DIAGNOSIS

Prenatal diagnosis must be considered in the contest of

Fig.2. Commonly used pedigree symbols

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the entire situation – the risk of disease in the baby,confirmed diagnosis in affected child/carrier status inparents, availability of treatment for the disease in questionand above all the wish of the couple. Ideally the discussionand planning should start pre-pregnancy which isinvariably not the case in our situation. Prenatal diagnostictechniques are shown in Table 1 & 2.

Prenatal screening for common chromosomaldisorders has good sensitivity using maternal serum

biochemical markers and ultrasonography.

Maternal serum screening should be a routineprenatal test to determine the risk of anenploidies andcertain malformations like neural tube defects. Earlier,maternal serum screening was classically done in thesecond trimester but now first trimester screen has beenfound to be more effective and useful.

Second trimester screen can be done between 15-22weeks of pregnancy, however, it is best preferred at 16 -18

Table 1. Prenatal Screening/ Diagnostic Techniques

(i) Noninvasive techniques : Timing(a) Maternal serum screening– First trimester (PAPP-A& free BetaHCG) 11-13+6wk– Second Trimester

Triple test (AFP, HCG, unconjugated estriol) 16-18weeksQuadruple screen (AFP, HCG, unconjugated estriol &inhibinA) 16-18weeks

(b) Fetal inspection by– Fetal ultrasonography (USG)

First trimester (NT& Newer markers) 11-13+6wkSecond Trimester (Anomaly scan) 18-20weeks

– Fetal echocardiography 18-20weeks– Fetal MRI >26weeks

(ii) Invasive techniques– Chorionic villus sampling (CVS) 11-13weeks– Amniocentesis 16-18weeks– Cord blood sampling >18weeks– Fetal skin, liver or muscle biopsy 18-20weeks

Table 2 Compares various invasive techniques

Procedure Risk Timings Indications*

Amniocentesis Fetal loss 0.5-1% 16-18 weeks CytogeneticAmniotic fluid leak MolecularRespiratory problem Biochemical

TORCH infectionsCVS Fetal loss 1.5-2% 11-13 weeks Molecular

Fetomaternal hemorrhage Biochemical CytogeneticCordocentsis Fetal loss 2-3% after 18 weeks Hematological

InfectionsCytogeneticsMolecular

* Any of the samples obtained by fetal sampling can be used for cytogenetic, molecular or biochemical tests but CVSsample is a desired sample for DNA based tests where as amniotic fluid is preferred for cytogenetic analysis.

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weeks. Commonly used 2nd trimester markers are maternalserum alphafetoprotein (MSAFP), human Chorionicgonadotropin (hCG) and estriol (the triple screen). MSAFPand estriol is low and hCG is high in the maternal serum ifthe mother is carrying a Down syndrome fetus. A positivescreen is when the risk of Down syndrome is 1 in 250(which means if 250 women have the given screenparameters one would have a baby with Down syndrome).

High MSAFP (>2.5 MoM) can identify over 95% ofanencephaly and 75% of open spina bifida cases. Use ofthree markers for Down syndrome screening will give amaximum detection rate of around 70%. This approach willalso result in detection of approx. 50 % of al cases oftrisomy 18.

Quadruple Test

Addition of a 4th biochemical marker, Inhibin-A,(increased in DS pregnancies) in the second trimesterscreen, increases the sensitivity of screening for DS from60% to 75%.

First Trimester Screening

Although many markers have been studied in the firsttrimester two robust markers suggested are free hCG andpregnancy associated plasma protein A (PAPP-A). Free

hCG has been found to be elevated with the median MoMvalues of 2.15, almost similar to the second trimester.PAPP-A values are low with the median MoM of around0.45-0.51, this alteration is not seen in the second trimester.Based on the available data the detection rates using thesetwo markers varies between 60-67% with a false positiverate of 5%. Detection of trisomy 18 and 13 has also beenreported by first trimester screening with good detectionrates.

Values of these biochemical tests are to be interpreted inmultiples of median (MoM). Each lab has its own cut offsand the risk is calculated based on previous history, age,gestation, number of fetuses, smoking, weight, ethnicity,gravidity and parity, previous screening results assistedreproduction, pregnancy complications and diabetes (lowerlevels).

When first trimester biochemical screen is combinedwith nuchal translucency (combined test), detection rate fortrisomy 21 increases to 85% at a false positive rate of 5%.Inclusion of various other newer markers such as nasalbone, tricuspid regurgitation and ductus venosus furtherraises the sensitivity to 92% at a false positive rate of 5%.So first trimester combined screen is clearly has moresensitivity and specificity and provides earlyreassurance [6].

While offering any screening method, one should offerboth pretest as well as posttest counseling. One shouldremember that it is not a diagnostic test but is a screeningtest to pick up high risk pregnancies and is certainly not asubstitute for fetal karyotyping. Definitive diagnosis can beprovided by chromosomal studies on amniotic fluid,chorionic villus biopsy or cord blood sample.

Ultrasound scanning in prenatal diagnosis [7]

Fetal anomaly scan is done at 11-14 weeks and 18-20weeks to look at the major malformations and soft markers.

1st trimester scan

It has been shown that around this time there is strongassociation between chromosomal abnormality andabnormal accumulation of fluid behind baby’s neck,referred to as increased ‘fetal nuchal translucency.’ Thisapplies both to DS and other autosomal trisomy syndromeslike trisomy 13 and 18. By combining information onmaternal age with results of fetal nuchal translucency andthickness measurements, it is possible to detect approx.80% of fetuses with trisomy 21, if invasive testing is offeredto the 5 % of pregnant women with the highest risk.

2nd trimester scan

Significant sonographic findings are seen in nearly allfetuses with trisomy 13, 77-100% of trisomy 18. Currentsonographic criteria can identify 65%-75% of fetuses withDown syndrome with a false positive rate of 4-15% insecond trimester. Presence of multiple abnormalities raisesthe risk of any chromosomal abnormality to 35%. With thecombined usage of sonographic markers for Downsyndrome and maternal serum screening, the vast majorityof fetuses with Down syndrome could be potentiallydetected.

Perinatal pathology

In case of unexplained fetal deaths or detection of majorcongenital malformations on antenatal ultrasound, fetalautopsy for complete genetic evaluation is of utmostimportance in order to make a specific diagnosis andascertain the risk of recurrence.

CONCLUSION

Pediatricians and Gynaecologists are the primaryphysicians for the diagnosis, and management of childrenand high risk couples with genetic disorders. Also besidestreating the patients, physicians should make the parents orcouple aware of the genetic disorder, risk of recurrence,prognosis and prenatal diagnosis. The development ofgenetic and molecular biology methods has opened up new

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opportunities in genetic prenatal diagnosis. Geneticcounseling in association with modern prenatal diagnosticprocedures constitutes a basic element of prevention ofcongenital anomalies and genetic disorders.

REFERENCES

1. National Center for Biotechnology Information. OnlineMendelian Inheritance in Man (OMIM). Available at:www.ncbi.nlm.nih.gov/ omim/. Accessibility verified May21, 2003.

2. Peltonen L, McKusick VA. Genomics and medicine:dissecting human disease in the postgenomic era.Science. 2001; 291:1224-1229.

3. Nussbaum RL, McInnes RR, Williard HF. Genetic

APPENDIX-A

HOW TO SEND SAMPLES TO A GENETIC LAB

Genetic lab/Geneticist should be informed before sending the sample.PRENATAL SAMPLESAmniotic fluid sample-About 20 mL of clear amniotic fluid is sent in sterile tubes (tubes are collected from the lab). It isrequired for chromosomal analysis, DNA and biochemical analysis.#Chorionic villi sample- About 20 -30 mg of chorionic villi should be sent in the transport media. (It can be collected from thegenetic lab).It is required for DNA analysis, enzyme analysis and chromosomal analysis.##in the order of preferencePOST NATAL sampleBloodDNA studies: Collect 3-6 mL blood in EDTA (purple top vacutainer)Chromosomal analysis: Collect 3 mL of blood in heparin (green top vacutainer)Enzyme analysis: 3-6 mL blood in heparin(green top vacutainer)Product of conception/ Skin for chromosomal analysis or DNA can be sent in the culture media (can be collected from thegenetic lab) or normal saline with 2 drops of crystalline penicillin and gentamycin.

counseling and risk assessment. In Thompson andThompson Genetics in Medicine. 6th edition by WBSaunders 2001.

4. Harper PS.Prenatal diagnosis and related reproductiveaspects. In Practical Genetic counseling, 6th Edition,Arnold Publishers 2004.

5. Muller R F, Young ID. Genetic counseling. In Emery’sElements of Medical Genetics. Eleventh edition byChurchill Livingstone in 2002.

6. Malone FD, et al. FASTER research Consortium.N Engl JMed 2005; 353:2001-2011.

7. Shipp TD, Benacerraf BR. Second trimester ultrasoundscreening for chromosomal abnormalities. Prenataldiagnosis 2002; 22: 296-307.

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