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SLE

• HLA class I, class II, and class III are associated with SLE, but only classes I and II contribute independently to increased risk of SLE

• Other genes which contain risk variants for SLE are IRF5, PTPN22, STAT4, CDKN1A, ITGAM, BLK, TNFSF4 and BANK1. Some of the susceptibility genes may be population specific

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screening• Firstly, there is screening of individuals and couples

known to be at significant or high risk because of a positive family history-sometimes referred to as targeted, or family, screening because it focuses on those most likely to benefit. This includes carrier, or heterozygote, screening, as well as presymptomatic testing.

• Secondly, there is the screening offered to the general population, who are at low risk-this is sometimes referred to as community genetics.

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Screening

• Population screening involves the offer of genetic testing on an equitable basis to all relevant individuals in a defined population.

• Its primary objective is to enhance autonomy by enabling individuals to be better informed about genetic risks and reproductive options.

• A secondary goal is the prevention of morbidity due to genetic disease and alleviation of the suffering that this would impose.

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SCREENING THOSE AT HIGH RISK

• If it was easy to recognize carriers of autosomal and X-linked recessive disorders and persons who are heterozygous for autosomal dominant disorders which show reduced penetrance or a late age of onset, much doubt and uncertainty would be removed when providing information in genetic counseling.

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CARRIER TESTING FOR AUTOSOMAL RECESSIVE AND X-LINKED DISORDERS

• In a number of autosomal recessive disorders, such as some of the inborn errors of metabolism, e.g. Tay-Sachs disease, and the hemoglobinopathies, e.g. sickle-cell disease, carriers can be recognized with a high degree of certainty using biochemical or hematological techniques such that DNA analysis is not necessary.

• In other single-gene disorders, it is possible to detect or confirm carrier status by biochemical means in only a proportion of carriers, e.g. the presence of abnormal coagulation studies in a woman at risk of being a carrier for hemophilia.

• A significant proportion of obligate carriers of hemophilia will have normal coagulation, however, so that a normal result in a woman at risk does not exclude her from being a carrier.

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Occasionally, carriers for certain disorders can have mild clinical manifestations of the disease

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BIOCHEMICAL ABNORMALITIES IN CARRIERS

• In some disorders the biochemical abnormality seen is a direct product of the gene and the carrier status can be tested for with confidence. For example, in carriers of Tay-Sachs disease the range of enzyme activity (hexosaminidase) is intermediate between levels found in normal and affected persons.

• In many inborn errors of metabolism, however, the enzyme activity levels in carriers overlap with the normal range, so that it is not possible to reliably distinguish between heterozygote carriers and those who are homozygous normal.

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Indirect

• A grossly elevated serum creatine kinase (CK) often confirms the diagnosis of DMD in a boy presenting with features of the disorder.

• Obligate female carriers of DMD have, on average, serum CK levels that are elevated compared to those of the general female population. There is, however, a substantial overlap of CK values between normal and obligate carrier females.

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Random inactivation of the X chromosome

• There is another reason for difficulty with carrier testing in the case of X-linked recessive disorders. Random inactivation of the X chromosome in females means that many, often the majority, of female carriers of X-linked disorders cannot be reliably detected by biochemical methods.

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PRESYMPTOMATIC DIAGNOSIS OF AUTOSOMAL DOMINANT DISORDERS

• Many autosomal dominant single-gene disorders either have a delayed age of onset or exhibit reduced penetrance.

• The results of clinical examination, specialist investigations, biochemical studies and family DNA studies can allow one to predict whether a person has inherited the gene before the onset of symptoms or signs. This is known as presymptomatic diagnosis or predictive testing

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CLINICAL EXAMINATION • It is not unusual to examine an apparently unaffected relative of

someone with NF1 who has had no medical problems, only to discover that they have sufficient numbers of a diagnostic feature, such as café-au-lait spots or cutaneous neurofibromas, to confirm that they are affected. However, NF1 is a relatively rare example of a dominantly inherited disorder that is virtually 100% penetrant by the age of 5 or 6 years, with visible external features. With many other disorders clinical examination presents greater challenges.

• Reaching a diagnosis in Marfan syndrome can be notoriously difficult because of the variable features and the overlap with other joint hypermobility disorders, even though very detailed diagnostic criteria have been established.

• Poly cystic kidney• Tuberous sclerosis

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• In TSC imaging studies of the brain by CT scan to look for intracranial calcification is a more or less routine investigation, as well renal ultrasound to identify the cysts known as angiomyolipoma(ta). Use of these relatively non-invasive tests in relatives of persons with TSC can detect evidence of the condition in asymptomatic persons.

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SPECIALIST INVESTIGATION (Intracranial calcification (arrowed) in an asymptomatic person with tuberous sclerosis

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• It is important to point out, however, that the absence of these findings on clinical or specialist investigation does not always exclude the diagnosis of the disorder being tested for but does reduce the likelihood of the person concerned having inherited the gene.

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BIOCHEMICAL TESTS

• In a number of autosomal dominant disorders biochemical tests can determine whether or not a person at risk has inherited a gene. Examples include the use of serum cholesterol levels in persons at risk for familial hypercholesterolemia and assay of the appropriate urinary porphyrins or the specific enzyme deficiency in the various dominant porphyrias

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Autosomal disorders that show a delayed age of onset or exhibit reduced penetrance in which linked DNA markers or specific mutational analysis can be used to offer presymptomatic diagnosis

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Familial adenomatous polyposis

• in persons at risk for familial adenomatous polyposis, colonoscopy looking for the presence of colonic polyps can be offered as a regular screening procedure to those who have been shown to be at high risk of developing colonic cancer by molecular studies. Conversely, individuals who have been shown not to have inherited a mutation in the APC gene do not need to be screened.

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• In contrast, for persons at risk for HD, in which there is not yet any effective treatment to delay the onset or progression of the disorder, the benefit of predictive testing is not immediately obvious.

• It is possible that employers, life insurance companies and society in general will put indirect and, on occasion, direct pressure on persons at risk for inherited disorders to have such testing.

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POPULATION SCREENING

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CRITERIA FOR A SCREENING PROGRAM

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Sensitivity: a / (a + c)-proportion of true positives; specificity: d / (d + b)-proportion of

true negatives

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Sensitivity: 96 / (96 + 4) = 96%; specificity is 510 100 / (510 100 + 4980) = 99%∼

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• Table 20.5 explains this further. Of great interest too is the positive predictive value of a screening test, which is the proportion of positive tests that are true positives, and this is illustrated in Table 20.6.

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Newborn screening programs

• Newborn screening programs have been introduced on a widespread basis for phenylketonuria, galactosemia and congenital hypothyroidism.

• In all of these disorders early treatment can prevent the development of learning disabilities.

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• The case of newborn screening for Duchenne muscular dystrophy also deviates from the screening paradigm because there is no effective treatment.

• In this situation the indication is to try to identify families for whom genetic counseling could be offered with a view to alerting relevant females to their possible carrier status.

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PHENYLKETONURIA • Routine biochemical screening of newborn infants for

phenylketonuria was recommended by the Ministry of Health in the UK in 1969 after it had been shown that a low-phenylalanine diet could prevent the severe learning disabilities that previously had been a hallmark of this condition

• The screening test, which is sometimes known as the Guthrie test, is carried out on a small sample of blood obtained by heel-prick at age 7 days.

• An abnormal test result is further investigated by repeat analysis of phenylalanine levels in a venous blood sample. A low-phenylalanine diet is extremely effective in preventing learning

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Newborn screening programs

• . Any woman with phenylketonuria who is contemplating pregnancy should adhere to a strict low-phenylalanine diet both before and during pregnancy to minimize the risk of brain damage to her unborn child

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GALACTOSEMIA

• Classic galactosemia affects approximately 1 in 50 000 newborn infants and usually presents with vomiting, lethargy and severe metabolic collapse within the first 2 or 3 weeks of life.

• Newborn screening is based on a modification of the Guthrie test with subsequent confirmation by specific enzyme assay. The early introduction of appropriate dietary restriction can prevent the development of serious complications such as cataracts, liver failure and learning disabilities.

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CONGENITAL HYPOTHYROIDISM

• Screening for congenital hypothyroidism was first introduced in the USA in 1974 and is now undertaken in most parts of the developed world.

• treatment with life-long thyroxine replacement is extremely effective in preventing the severe developmental problems associated with the classic picture of 'cretinism'. The most common cause of congenital hypothyroidism is absence of the thyroid gland rather than an inborn error of metabolism

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CYSTIC FIBROSIS

• It is based on the detection of an elevated blood level of immunoreactive trypsin (IRT), which is a consequence of blockage of pancreatic ducts in utero, supplemented by DNA analysis

• The rationale for screening is that it is hoped that early treatment with physiotherapy and antibiotics will improve the long-term prognosis.

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SICKLE-CELL DISEASE AND THALASSEMIA

• Newborn screening based on hemoglobin electrophoresis is undertaken in many countries with a significant Afro-Caribbean community.

• . In the case of sickle-cell disease, treatment involves the use of oral penicillin to reduce the risk of pneumococcal infection resulting from immune deficiency secondary to splenic infarction

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POPULATION CARRIER SCREENING

• Widespread screening for carriers of autosomal recessive disorders in high-incidence populations was first introduced for the hemoglobinopathies and has been extended to several other disorders.

• The rationale behind these programs is that carrier detection can be supported by genetic counseling so that carrier couples can be forewarned of the 1 in 4 risk that each of their children could be affected.

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POPULATION CARRIER SCREENING

• In Cyprus in 1974 the birth incidence of β-thalassemia was 1 in 250 (carrier frequency 1 in 8). Following the introduction of a comprehensive screening program to determine the carrier status of young adults, which had the support of the Greek Orthodox Church, the incidence of affected babies declined by over 95% within 10 years

• Similar programs in Greece and Italy have seen a drop in the incidence of affected homozygotes of over 50%.

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POSITIVE AND NEGATIVE ASPECTS OF POPULATION SCREENING

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TECHNIQUES USED IN PRENATAL DIAGNOSIS

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Amniocentesis

• Amniocentesis involves the aspiration of 10-20ml of amniotic fluid through the abdominal wall under ultrasound guidance

• This is usually performed around the 16th week of gestation. The sample is spun down to yield a pellet of cells and supernatant fluid.

• The fluid can be used in the prenatal diagnosis of neural tube defects by assay of α-fetoprotein

• The availability of PCR has meant that direct DNA analysis is usually possible without the need for culture.

• When a couple is considering amniocentesis as an option they should be informed of the 0.5-1% risk of miscarriage associated with the procedure

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A diagram of the technique of amniocentesis

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CHORIONIC VILLUS SAMPLING

• In contrast to amniocentesis, chorionic villus sampling (CVS), which was first developed in China, enables prenatal diagnosis to be undertaken during the first trimester.

• This procedure is usually carried out at 11-12 weeks gestation under ultrasound guidance by either transcervical or, more usually, transabdominal aspiration of chorionic villi

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• Chromosome analysis can be undertaken on chorionic villi either directly, looking at metaphase spreads from actively dividing cells, or following culture.

• Direct chromosomal analysis of chorionic villi usually allows a provisional result to be given within 24h.

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CVS

• although it has the disadvantage that even in experienced hands this procedure conveys a 1-2% risk of causing miscarriage.

• There is also evidence that this technique can cause limb abnormalities in the embryo if carried out before 9-10 weeks gestation - for this reason CVS is not now performed before 11 weeks gestation.

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CHORIONIC VILLUS SAMPLING

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ULTRASOUND

• ULTRASOUND can be used not only for obstetric indications, such as placental localization and the diagnosis of multiple pregnancies, but also for the prenatal diagnosis of structural abnormalities that are not associated with known chromosomal, biochemical or molecular defects.

• For example, a search can be made for polydactyly as a diagnostic feature of a multiple abnormality syndrome, such as one of the autosomal recessive short-limb polydactyly syndromes that are associated with severe pulmonary hypoplasia and are invariably lethal. Similarly,

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polydactyly

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Micrognathia (small jaw)

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• however, detailed 'fetal anomaly' scanning is being offered routinely to all pregnant women at around 18 weeks gestation as a screening procedure for structural abnormalities such as neural tube defects or cardiac anomalies.

• In addition, the observation that increased nuchal translucency (NT) is seen in fetuses who are subsequently born with Down syndrome, has resulted in the introduction of measurements of nuchal pad thickness in the first and second trimesters as a screening test for Down syndrome

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FETOSCOPY

• Fetoscopy involves visualization of the fetus by means of an endoscope. Increasingly, this technique is being superseded by detailed ultrasound scanning, although occasionally fetoscopy is still undertaken during the second trimester to try to detect the presence of subtle structural abnormalities that would point to a serious underlying diagnosis.

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• Fetoscopy has also been used to obtain samples of tissue from the fetus that can be analyzed as a means of achieving the prenatal diagnosis of several rare disorders like epidermolysis bullosa (skin) and ornithine transcarbamylase deficiency (liver)

• Unfortunately, fetoscopy is associated with a 3-5% risk of miscarriage.

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CORDOCENTESIS

• Although fetoscopy can also be used to obtain a small sample of fetal blood from one of the umbilical cord vessels in the procedure known as cordocentesis, improvements in ultrasound have enabled visualization of the vessels in the umbilical cord, allowing transabdominal percutaneous fetal blood sampling.

• Fetal blood sampling is routinely used in the management of rhesus isoim-munization and can be used to obtain samples for chromosome analysis to resolve problems associated with possible chromosomal mosaicism in chorionic villus or amniocentesis samples.

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RADIOGRAPHY

• The fetal skeleton can be visualized by radiography from 10 weeks onwards and this technique has been used in the past to diagnose inherited skeletal dysplasias.

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PRENATAL SCREENING

• MATERNAL SERUM SCREENING • maternal serum screening is offered for neural

tube defects and Down syndrome using a blood sample obtained from the mother at 16 weeks gestation. In this way up to 75% of all cases of open neural tube defects and 60-70% of all cases of Down syndrome can be detected.

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NEURAL TUBE DEFECTS

• In 1972 it was recognized that many pregnancies in which the baby had an open neural tube defect (NTD) could be detected at 16 weeks' gestation by assay of a protein in maternal serum known as α-fetoprotein (αFP). αFP is the fetal equivalent of albumin and is the major protein in fetal blood.

• If the fetus has an open NTD, the level of αFP is elevated in both the amniotic fluid and maternal serum as a result of leakage from the open defect. Open NTDs fulfil the criterion of being serious disorders, as anencephaly is invariably fatal,

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• The curves for the levels of maternal serum αFP in normal and affected pregnancies overlap, so that in practice an arbitrary cut-off level has to be introduced below which no further action is taken.

• This is usually either the 95th centile or 2.5 multiples of the median (MoM), and as a result around 75% of screened open spina bifida cases are detected.

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Women with a value on or above 2.5 multiples of the median are offered further investigations

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Causes of elevated maternal serum αFP

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• Other contributory factors are a general improvement in diet and the introduction of periconceptional folic acid supplementation

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Maternal risk factors for Down syndrome

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• This latter approach is based on the discovery that, at 16 weeks gestation, maternal serum αFP and unconjugated estriol levels tend to be lower in Down syndrome pregnancies than in normal pregnancies, whereas the level of maternal serum human chorionic gonadotropin is usually elevated. None of these parameters gives absolute discrimination, but taken together they provide a means of modifying a woman's prior age-related risk to give an overall probability that the unborn baby is affected.

• When this probability exceeds 1 in 250, invasive testing in the form of amniocentesis or placental biopsy is offered.

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• In trisomy 18 all the biochemical parameters are low, including hCG.

• It has recently been shown that another biochemical marker, inhibin-A, is also increased in maternal serum in Down syndrome pregnancies. Quadruple test

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Ultrasonography

• Almost all pregnant women are routinely offered a 'dating' scan at around 12 weeks gestation

• Nuchal translucency (NT) applies to Down syndrome, the other autosomal trisomy syndromes, i.e. trisomy 13 and trisomy 18, Turner syndrome, triploidy, as well as a wide range of other fetal abnormalities and rare syndromes.

• The risk of Down syndrome correlates with absolute values of NT as well as maternal age but, as NT also increases with gestational age, it more usual now to relate the risk to the centile value for any given gestational age. In one study, 80% of Down syndrome fetuses had NT above the 95th centile.

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• A chromosome abnormality is found in 50% of fetuses with an exomphalos identified at 18 weeks, and a rocker-bottom foot is a very characteristic finding in babies with trisomy 18, who are also invariably growth retarded.

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ADVANCED MATERNAL AGE

• Most centers routinely offer amniocentesis or CVS to women aged 37 years or over, and the option is often discussed with women from the age of 35 years onwards.

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PREVIOUS CHILD WITH A CHROMOSOME ABNORMALITY

• recurrence risk figures, for couples who have had a child with Down syndrome due to non-disjunction or a de novo unbalanced Robertsonian translocation, the risk in a subsequent pregnancy is usually given as the mother's age-related risk plus approximately 0.5%.

• If one of the parents has been found to carry a balanced chromosomal rearrangement, such as a chromosomal translocation or pericentric inversion, which has caused a previous child to be born with serious problems due to an unbalanced chromosome abnormality, then the recurrence risk is likely to be between 1-2% and 15-20%.

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FAMILY HISTORY OF A NEURAL TUBE DEFECT

• In high-risk situations amniocentesis with assay of the chemical α-fetoprotein has been used for prenatal diagnosis in the past. Ultrasound examination of the fetus in conjunction with assay of maternal serum α-fetoprotein has proved equally reliable.

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FAMILY HISTORY OF OTHER CONGENITAL STRUCTURAL ABNORMALITIES

• If the risk to a pregnancy is increased, detailed ultrasound examination looking for the specific structural abnormality can be offered at around 16-18 weeks gestation. Mid-trimester ultrasound will detect most serious cranial, cardiac, renal and limb malformations.

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OTHER HIGH-RISK FACTORS

• These include parental consanguinity, a poor obstetric history and certain maternal illnesses.

• . A poor obstetric history, such as recurrent miscarriages or a previous unexplained stillbirth, could indicate an increased risk of problems in a future pregnancy and would be an indication for detailed ultrasound monitoring.

• A history of three or more unexplained miscarriages should be investigated by parental chromosome studies to exclude a chromosomal rearrangement such as a translocation or inversion

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• Maternal illnesses, such as poorly controlled insulin-dependent diabetes mellitus or epilepsy treated with anticonvulsant medications such as sodium valproate, would also be indications for detailed ultrasonography.

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SPECIAL PROBLEMS IN PRENATAL DIAGNOSIS

• FAILURE TO OBTAIN A SAMPLE OR CULTURE FAILURE Fortunately, the risk of either of these events

occurring is less than 1%• AN AMBIGUOUS CHROMOSOME RESULT• In approximately 1% of cases, CVS shows evidence

of apparent chromosome mosaicism, i.e. the presence of two or more cell lines with different chromosome constitutions. This can occur for several reasons:

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• The sample is contaminated by maternal cells.• culture artifact• If mosaicism is present in only one culture then it is

probably an artifact and does not reflect the true fetal karyotype.

• The mosaicism is limited to a portion of the placenta, or what is known as confined placental mosaicism.

• The mosaicism is limited to a portion of the placenta, or what is known as confined placental mosaicism.

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3 levels of mosaicism

• If a single abnormal cell is identified in only one culture this is assumed to be a culture artifact, or what is termed level 1 mosaicism (pseudomosaicism)

• If the mosaicism extends to two or more cells in two or more cultures this is taken as evidence of true mosaicism, or what is known as level 3 mosaicism

• The most difficult situation to interpret is when mosaicism is present in two or more cells in only one culture, which is termed level 2 mosaicism.

• There is up to a 20% chance that the mosaicism is real and will be present in the fetus

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The presence of a marker chromosome

• marker chromosome, is a small chromosomal fragment the specific identity of which cannot be determined by conventional cytogenetic techniques

• If this is found to be present in one of the parents, then it is unlikely that it will be of any significance to the fetus.

• If, on the other hand, it is a de novo finding, there is up to a 15% chance that the fetus will be phenotypically abnormal.

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Marker chromosome

• The risk is lower if the marker chromosome contains satellite material, or is largely made up of heterochromatin

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ULTRASOUND 'SOFT' MARKERS

• choroid plexus cysts are sometimes seen in the developing cerebral ventricles in mid-trimester. Initially, it was thought that these were invariably associated with the fetus having trisomy 18.

• It is now known that choroid plexus cysts occur frequently in normal fetuses, although if they are very large and do not disappear spontaneously they can be indicative of a chromosome abnormality.

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TERMINATION OF PREGNANCY

• The presence of a serious abnormality in a fetus in the majority of developed countries is an acceptable legal indication for termination of pregnancy.

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PGD

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PGD

• In the procedure the female partner is given hormones to induce hyperovulation, and oocytes are then harvested transcervically, under sedation, with ultrasound guidance.

• Motile sperm from a semen sample are added to the oocytes in culture (in-vitro fertilization (IVF) - the same technique as developed for infertility) and incubated to allow fertilization to occur.

• If genetic analysis is to be undertaken on DNA from a single cell (blastomere) from the early embryo (blastocyst) at the eight-cell stage on the third day, fertilization is achieved using intracytoplasmic sperm injection (ICSI) of a single sperm to avoid the presence of extraneous sperm.

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PGD

• Choosing a desired sex (boy or girl)• Choosing a normal cell with matched HLA with

his or her affected siblings• Micromanipoulation: The nucleus of the

oocyte from the genetic mother (who carried the mitochondrial mutation) was removed and inserted into a donor oocyte from which the nucleus had been removed. This is cell nuclear replacement (CNR) technology.

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IVF and ICSI

• In these methods the risk of congenital abnormalities was basically the same as for the general population conceiving in the normal way.

• However, concern has begun to emerge that there might be a small but abnormal increase in the incidence of conditions due to defective genomic imprinting

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DONOR INSEMINATION

• screening of sperm donors for cystic fibrosis mutations and chromosome rearrangements has become routine practice in many countries

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DETECTION OF FETAL CELLS IN THE MATERNAL CIRCULATION

• Using antibodies raised to antigens specific to the fetal trophoblast, there is evidence that cells of fetal origin are present in the maternal circulation in the first trimester.

• For Rh compatibility (embryonic RBC)

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PRENATAL TREATMENT • A possible model for successful prenatal treatment is provided by

the autosomal recessive disorder congenital adrenal hyperplasia (CAH) .

• There is evidence that in a proportion of cases the virilization can be prevented if the mother takes a powerful steroid known as dexamethasone in a very small dose from 4 to 5 weeks gestation onwards.

• Specific prenatal diagnosis of CAH can be achieved by DNA analysis of chorionic villi. If this procedure confirms that the fetus is both female and affected, then the mother continues to take low doses of dexamethasone throughout pregnancy, which suppresses the fetal pituitary - adrenal axis and can prevent virilization of the female fetus.

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