Molecular genetics

Molecular genetics

Current Obstetrics & Gynaecology (2000) 10, 170–174 © 2000 Harcourt Publishers Ltd doi:10.1054/cuog.2000.0135, available online at http://www.idealibr...

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Current Obstetrics & Gynaecology (2000) 10, 170–174 © 2000 Harcourt Publishers Ltd doi:10.1054/cuog.2000.0135, available online at on

Self-assessment questions

Molecular genetics

A. McEwan

Question 1

Question 2

Miss FB is a 36 year-old woman (indicated by the arrow) who expresses a concern regarding her family history of breast and ovarian cancer. She is unmarried and has no children. After careful questioning you are able to elicit the family tree shown in Fig. 1 How would you advise FB regarding her own risks of developing breast or ovarian cancer? Which investigations might be offered and how might the case be subsequently managed?

Answer true of false. Regarding hereditary cancer syndromes: 1. BRCA2 mutations carry a greater lifetime risk of ovarian cancer than BRCA1 mutations. 2. Approximately 15% of all cases of breast cancer are due to germline mutations. 3. Cervical cancer is found within the spectrum of malignancies associated with the Li-Fraumeni syndrome. 4. Colorectal and endometrial cancers may ‘cluster’ within families. 5. Ashkenazi Jews are at greater risk of inherited breast cancer. Question 3 Answer true or false. Which of the following conditions/syndromes are associated with an increased risk of ovarian carcinoma? 1. 2. 3. 4. 5.

MSH6 germline mutations Turner’s syndrome (45X0) Lynch II HNPCC Testicular feminization syndrome p53 germline mutations

Question 4

Fig. 1

Answer true or false. In which of the following genes can mutations cause male infertility?

Alec McEwan, Clinical Research Fellow, Department of Obstetrics & Gynaecology, City Hospital, Hucknall Road, Nottingham NG5 1PB, UK. Tel: +44 (0)115 962 7923; E-mail: [email protected]

1. 2. 3. 4. 5.



Molecular genetics


Question 5

Question 9

Answer true or false.

Answer true or false. Regarding preimplantation genetic diagnosis (PGD):

1. Y chromosome microdeletions cause congenital absence of the vas deferens. 2. XXY syndrome causes male infertility. 3. All the genes responsible for spermatogenesis are found on the Y chromosome. 4. ICSI (intracytoplasmic sperm injection) protects offspring from inherited forms of male infertility. 5. Environmental factors are responsible for male infertility in the majority of cases. Question 6 Answer true or false. Regarding fetal cell isolation: 1. CD71 is a cell surface antigen which is exploited in the isolation of trophoblasts from maternal blood. 2. Sorting fetal cells from maternal blood for the diagnosis of trisomy 21 has a low false positive rate. 3. Single gene defects can be diagnosed using techniques adapted from preimplantation genetic diagnosis. 4. In a second trimester maternal blood sample, the majority of nucleated red blood cells are fetal in origin. 5. Large trials have validated the role of transcervical cell sampling in non-invasive prenatal diagnosis.

1. Outer trophoectodermal cells are removed from the embryo on day 3, prior to transfer. 2. Use of comparative genomic hybridization (CGH) is limited by time. 3. Multiplex PCR helps to reduce errors. 4. Pregnancy rates are significantly higher than those following IVF for infertile couples. 5. First polar body biopsy represents the earliest screening for the inheritance of maternal and paternal mutations. Question 10 Figure 2 shows an ultrasound scan performed at 18 weeks gestation in a 36 year-old woman. She agreed to have a peripheral blood sample taken for the isolation of fetal cells. Figure 3 shows one of these isolated fetal cells (a nucleated red blood cell). What abnormality is shown in Figure 2? Which conditions is it associated with? Which analytical genetic technique is demonstrated in Figure 3 and what do you think the most likely diagnosis is? How would you counsel her?

Question 7 Answer true or false. 1. The stability of Taq polymerase at high temperatures is crucial for the polymerase chain reaction (PCR) 2. Aneuploidy can be diagnosed from single fetal cells using PCR techniques. 3. Multiplex PCR analyses a large number of different samples in a short space of time. 4. Cell recycling increases the amount of information obtainable from the analysis of a single cell. 5. Fluorescent in-situ hybridization (FISH) is used to diagnose thalassaemias from isolated fetal cells.

Fig. 2

Question 8 Answer true or false. The following conditions have been tested for using preimplantation genetic diagnosis: 1. 2. 3. 4. 5.

Meckel syndrome VATER association Patau’s syndrome Lesch-Nyhan syndrome Familial adenomatous polyposis coli

Fig. 3


Current Obstetrics & Gynaecology

Answer 1 FB should be placed in high risk categories for the development of both breast and ovarian cancer. It is extremely likely that a germline mutation predisposing to cancer is being transmitted through this family in an autosomal dominant fashion, in which case FB would have a 50% chance of being a carrier. A mutation in BRCA1 is most likely. These carry overall breast and ovarian cancer risks of 40% and 44% respectively, by the age of 70 years (although in some families the risks can be significantly higher). FB’s own empiric risks are half these values as her carrier status is unclear. This family should be offered genetic counselling and mutation testing of BRCA1 and BRCA2. If affected family members are found to have a mutation then the others can be offered predictive testing. If FB tests negative then she still has a ‘background’ risk i.e. one in 12 lifetime risk for breast cancer and one in 80 for ovarian cancer. If she does not undergo mutation testing, or if she tests positive then she should be offered screening with 1–2 yearly mammograms and yearly transvaginal scans as a minimum. Currently, however, there is no good evidence that screening reduces mortality rates from ovarian cancer. A more radical management plan would be bilateral mastectomy and oophorectomy. In-depth counselling and preparation is vital and uncertainties still remain over ongoing risks of malignancy and the use of HRT in these women. Answer 2 1. False 2. False 3. False 4. True 5. True The cumulative risk for ovarian cancer in BRCA1 mutation carriers is 29% by age 50 and 44% by age 70. The equivalent risks for BRCA2 mutation carriers are much less (0.4% and 27% respectively). Approximately 5% of breast cancers are caused by germline mutations. Cancers typically found in the LiFraumeni syndrome are adrenocortical, breast, brain, sarcomas and leukaemia. There are no known inherited cancer syndromes which include cervical carcinoma. Endometrial cancers can associate with colorectal cancers in Lynch II families where inherited abnormalities of the DNA mismatch repair genes are common. One in 100 Ashkenazi Jews carry a BRCA1 mutation and one in 70 a BRCA2 mutation. In the general population approximately one in 900 have a germline mutation in one of these genes. Answer 3 1. True 2. False

3. True 4. False 5. False MSH6 is a DNA mismatch repair gene. Inherited defects within this gene can cause Hereditary nonpolyposis colon cancer (Lynch I or II). Lynch II families have an increased risk of developing cancers of the colon, rectum, endometrium, stomach, bladder, pancreas and ovary. Turnerís syndrome (45XO) causes streak ovaries that are not at increased risk of malignancy. Only the mosaic cases of Turner’s syndrome (45XO/46XY) carry the increased risk of gonadoblastoma and it is these individuals who should undergo gonadectomy. Patients with the testicular feminization syndrome have genetic defects within the androgen receptor gene. They have a 46XY karyotype and dysgenic testes which are predisposed to testicular, rather than ovarian, tumours (commonly seminomas). Germline p53 mutations cause the LiFraumeni syndrome and ovarian cancer does not feature within its array of associated malignancies.

Answer 4 1. True 2. False 3. True 4. False 5. False Mutations within the DAZ (deleted in azoospermia) gene severely impair spermatogenesis. Mutations of the CFTR (cystic fibrosis transmembrane conductance regulator) gene can result in obstructive azoospermia by causing congenital bilateral absence of the vas deferens. Mutations in the PKD1, SMN and MLH1 genes cause adult polycystic kidney disease (APKD), spinal muscular atrophy (SMA) and hereditary non-polyposis colorectal cancer (HNPCC) respectively. They are not associated with genetic male infertility. Answer 5 1. False 2. True 3. False 4. False 5. False Y chromosome microdeletions of the AZF (azoospermia factor) locus cause azoospermia or severe oligozoospermia as the genes in this region are critical for spermatogenesis. Congenital bilateral absence of the vas deferens (CBAVD) is most commonly caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), found on chromosome 7. Other autosomal genes must also contribute to spermatogenesis as reciprocal and Robertsonian

Molecular genetics translocations can cause male factor infertility. Klinefelter’s syndrome (XXY) is associated with hypogonadism with hyalinized seminiferous tubules and germ cell degeneration during puberty. ICSI enables men with abnormal sperm and extremely low counts to father offspring. There is significant concern that genetic defects causing the male infertility will be passed onto the sons of these men through the use of ICSI. Although there is no firm evidence yet that the offspring of ICSI have an increased risk of congenital abnormalities it will be a number of years before their reproductive capacity becomes evident. All men with CBAVD should be tested for CFTR mutations. If they are carriers then their partners should also be offered testing and counselled regarding the risks of cystic fibrosis in their ICSI offspring. Answer 6 1. False 2. True 3. True 4. False 5. False CD71 is the transferrin receptor and is found on the surface of cells actively incorporating iron. However, it is not expressed on trophoblasts and is used to sort fetal nucleated red cells from maternal blood. The ‘NIFTY’ trial, to date, is giving a 40–50% sensitivity for the detection of aneuploidies in high risk women. The false positive rate is low (<1%) as compared with the false positive rates associated with nuchal translucency scanning and second trimester biochemical screening, which are artificially set at approximately 5%. Micromanipulation of individual blastomeres was perfected for PGD. Similar techniques are now being used on fetal cells isolated from maternal blood to free them of contamination from maternal cells, so allowing complex DNA analysis for single gene defects. There have been no large trials to assess the role of transcervical cell sampling in routine prenatal diagnostic services. Answer 7 1. True 2. True 3. False 4. True 5. False Taq polymerase is a thermostable DNA polymerase originally extracted from a bacterium called Thermophilus aquaticus which grows in hot springs. The enzyme functions at 72°C and is stable up to 93°C. Quantitative PCR techniques, often employing fluorescently labelled primers or oligonucleotides, are able to diagnose aneuploidies such as trisomy 21 and 18 from single cells, providing an alternative to FISH.


Multiplex PCR is an advance on the original technique which employs a number of different primer pairs to replicate more than one genetic locus from the same sample. PCR conditions often have to be compromized, making this a difficult technique to optimize. It does however increase the number of analyses which can be performed on a very limited DNA source, such as a single isolated fetal cell. Cell recycling allows the sequential use of FISH followed by PCR on the same individual cell. Fluorescent in-situ hybridization (FISH) is used to detect abnormalities of chromosome number and not usually single gene defects, which are responsible for the thalassaemias. Answer 8 1. False 2. False 3. True 4. True 5. True Meckel syndrome is an autosomal recessive condition characterized by central nervous system malformations (typically occipital meningoencephalocoele), bilaterally large multicystic kidneys and polydactyly. Although a locus on chromosome 17 has been implicated in some cases the genetic defects responsible mostly remain to be elucidated. Prenatal diagnosis is usually carried out by transvaginal scanning at the end of the first trimester. The VATER association is of unknown aetiology and includes Vertebral anomalies, Anal atresia, Tracheoesophageal fistula, Esophageal atresia and Radial dysplasia. Cardiac and renal abnormalities are also common. Almost all cases are sporadic and the recurrence risk is very low, excluding a role for PGD. The risk of Patau’s syndrome (Trisomy 13) increases with maternal age. Most of these fetuses are lost as early miscarriages, however, a few survive to a viable gestation, the infants suffering from a number of very serious congenital anomalies which are not compatible with long-term survival. Using FISH techniques, aneuploidy can be screened for at the time of PGD and there is some evidence that testing all embryos for common aneuploidies prior to transfer may help increase implantation rates and reduce miscarriage risk in the older IVF patient. The Lesch-Nyhan syndrome is a severe metabolic disorder caused by the deficiency of hypoxanthine phosporibosyl transferase (HPRT). Being X-linked it was one of the first conditions to be screened for using PGD. Initially this consisted of sexing the embryos and transferring only females. Now, if the parental mutations have been identified, the mutation status of each embryo can be tested. Unaffected male embryos can be transferred, as well as female. Familial adenomatous polyposis coli is an autosomal dominant condition which causes multiple polyps to form throughout the large bowel. Mutation carriers will develop bowel cancer by 50–60 years of age and


Current Obstetrics & Gynaecology

prophylactic total colectomy is advised. Other cancers also occur with increased frequency. PGD has been successfully used to screen for this condition. Answer 9 1. False 2. True 3. True 4. False 5. False Most cases of PGD involve biopsy at the cleavage stage on day 3 when the embryo is a mere 6–10 cells (blastomeres) in total. Trophoectoderm cells can be removed from human blastocysts between days 5 and 8 and subsequently used for PGD. Comparative genomic hybridization (CGH) is a complex technique for comparing relative amounts of genetic information from two soures (one a control). When applied to single cells aneuploidy has been detected. All the chromosomes can be ‘counted’ using this analysis. Unfortunately it is time consuming and this limits its application in PGD where time from embryo biopsy to transfer is restricted. Multiplex PCR allows a number of different genetic loci to be studied in the same analysis. This reduces error rates in PGD by helping to detect allele drop out and amplification failure. Clinical pregnancy rates after PGD are similar to those resulting from IVF for infertility (22% and 24% per embryo transfer respectively). The first polar body can be sampled prior to fertilization occurring. This gives information regarding the maternal genetic information passed on but not the paternal.

Answer 10 The ultrasound picture shows an exomphalos (omphalocoele), a herniation of the intra-abdominal viscera through an open umbilical ring into the base of the umbilical cord. It is commonly found in cases of aneuploidy (typically trisomy 18, but also trisomy 13 and 21 occasionally) and is also one of the features of a number of different conditions, including Beckwith Wiedemann syndrome, Pentalogy of Cantrell and the CHARGE association. Figure 3 is a cell which has undergone a FISH procedure, demonstrating three fluorescent signals with the same probe. This suggests a trisomy and although no information is given regarding which FISH probe was used the most likely diagnosis is trisomy 18 (Edward’s syndrome). Indeed, as many as 40% of exomphalos cases have a chromosomal abnormality, the most common being trisomy 18. Fetal cell isolation from maternal blood is still undergoing assessment in a large multicentre trial. It is not yet used in everyday clinical practice. Although false positive results are very uncommon with this technique, making the diagnosis of trisomy 18 very likely, this woman should be offered an invasive test to confirm the result. Trisomy 18 is associated with severe neurological and cardiac abnormalities and the affected children do not survive for long postnatally. Indeed many will die in utero if the pregnancy is not terminated. If the diagnosis is confirmed then offer of termination should be made. Although most couples will accept this offer some will opt to continue the pregnancy. They need ongoing antenatal care but in a sensitive environment away from the normal antenatal clinic.