MOLECULAR GENETICS - INDIVIDUAL GENETIC DISORDERS

---

Studies should be performed in association with counselling and by prior arrangement with the laboratory - consult pathologist. If other family members are known to be affected and/or have had molecular genetic studies performed, this information should be provided to the laboratory. The diseases listed represent a limited selection of diseases for which molecular genetic diagnoses are available. Consult your Pathologist for further advice.

CHARCOT-MARIE-TOOTH DISEASE

Specimen: Check with laboratory.

Method: Southern blot analysis to detect the common mutations within the peripheral myelin protein 22 (PMP22) gene responsible for Type IA Charcot-Marie-Tooth disease and tomaculous neuropathy.

Application: Used in conjunction with neurophysiological studies to diagnose Type IA Charcot-Marie-Tooth disease and tomaculous neuropathy. No tests are available for Type II (axonal) Charcot-Marie-Tooth disease.

Interpretation: Current laboratory methods detect approximately 60% of the PMP22 mutations responsible for tomaculous neuropathy. The identification of a duplication of the PMP22 gene is diagnostic of Type IA Charcot-Marie-Tooth disease. A deletion within the gene is diagnostic of tomaculous neuropathy. The identification of a mutation does not predict the age of onset or severity. Molecular genetic studies may be indicated in other family members. The absence of a mutation does not exclude the diagnosis and family studies may clarify a person’s carrier status.

Reference: Nicholson G. J Neurol Neurosurg Psychiatry 1995; 58: 523-525.

CYSTIC FIBROSIS

Specimen: Dried blood spot on neonatal screening card; 2-10 mL blood in EDTA tube; 4 plucked hair roots or buccal smear/mouth wash.

Method: PCR-based analysis to detect a selection of common mutations in the CFTR gene.

Application: Used in conjunction with sweat chloride in the diagnosis of cystic fibrosis in neonates identified by a high trypsinogen at neonatal screening, children or adults with clinical features suggesting cystic fibrosis, incl bronchiectasis and congenital bilateral absence of vas deferens. Used to define carrier status in those with a relative who has cystic fibrosis or who has been shown to be a carrier. Prenatal diagnosis for cystic fibrosis is available if both parents have identified mutations or if DNA polymorphism studies allow identification of both abnormal chromosomes in the family. See also CHLORIDE - sweat and NEONATAL SCREEN .

Interpretation: The number and type of mutations tested varies between laboratories, but the mutations surveyed account for 70-80% of all mutations responsible for cystic fibrosis. The most common mutation is delta F 508, accounting for about 70% of mutations. About 60% of patients with cystic fibrosis will have two mutations detected, 35% will have one mutation detected and 5% will have no mutation detected. The identification of two pathogenic mutations is diagnostic of cystic fibrosis; the identification of one mutation in an asymptomatic person with normal sweat chloride is diagnostic of the carrier status for cystic fibrosis. Molecular genetic studies may be indicated in other family members. These methods do not detect all possible mutations and the lack of defined mutations does not necessarily exclude the diagnosis of carrier status or cystic fibrosis. Family studies, involving DNA polymorphism, may clarify an individual’s carrier status.

Reference: Danks DM. Med J Aust 1993; 159: 148-150.

DUCHENNE/BECKER MUSCULAR DYSTROPHY

Specimen: 10 mL blood in EDTA tube.

Method: Southern blot or PCR-based analysis to detect a deletion in the dystrophin gene.

Application: Used in conjunction with creatine kinase in the diagnosis of Duchenne/Becker muscular dystrophy and in identifying female carriers. Prenatal diagnosis for Duchenne muscular dystrophy may be available if the mother has been shown to be a carrier.

Interpretation: Deletions within the dystrophin gene account for approximately 65% of cases of Duchenne/Becker muscular dystrophy in boys. These deletions can usually be identified in female carriers. The identification of a deletion in the dystrophin gene in a symptomatic male is diagnostic of Duchenne/Becker muscular dystrophy. The identification of a deletion in one dystrophin gene in a female is diagnostic of the carrier status. Molecular genetic studies may be indicated in other family members. The absence of a deletion does not exclude the diagnosis and family studies may clarify a woman’s carrier status.

Reference: Laing NG et al. Med J Aust 1991; 154: 14-18.

FACTOR V LEIDEN MUTATION

Specimen: 5-10 mL blood in EDTA tube.

Method: PCR-based analysis.

Application: Aids in the investigation of thrombophilia of possible congenital origin.

Interpretation: The majority (>90%) of patients with reduced activated protein C resistance have an inherited disorder of the (coagulation) factor V molecule (Arg506Gln) and are at increased risk of venous thromboembolism. The disorder is characterised by a low anticoagulant response of plasma on addition of activated protein C (see ACTIVATED PROTEIN C (APC) RESISTANCE TEST ). The abnormality has been reported in 20-60% of patients with venous thromboembolism, although thrombosis usually only occurs when other risk factors are present.

Reference: Svesson PJ and Dahlbäck B. N Engl J Med 1994; 330: 517-522. Dahlbäck B. Thromb Haemost 1995; 73: 739-742. Bovill EG et al. Thromb Haemost 1999; 82: 662.

FAMILIAL POLYPOSIS COLI

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analysis to detect the presence of common mutations within the APC (adenomatous polyposis coli) gene.

Application: Used in conjunction with colonoscopy to diagnose familial polyposis coli. Used to identify asymptomatic carriers.

Interpretation: Current laboratory methods detect 70-80% of the mutations in the APC gene. The identification of a mutation within the APC gene is diagnostic for familial polyposis coli. The identification of a mutation does not predict the likely age of onset of colon cancer or the possibility of significant extra-colonic complications. Molecular genetic studies are indicated in other family members. The absence of a mutation does not exclude the diagnosis and family studies may clarify a person’s carrier status.

Reference: Walpole IR et al. Med J Aust 1995; 162: 464-467. Gardner M and St John J. Med J Aust 1995; 162: 457.

FRAGILE X SYNDROME

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analysis to detect an expanded triplet repeat mutation in the regulatory region of the FMR-1 (fragile-site mental retardation) gene.

Application: Investigation of familial or sporadic mental retardation without obvious cause, in conjunction with cytogenetics, to detect the fragile X mutation. Prenatal diagnosis for the fragile X syndrome is available; consult pathologist.

Interpretation: In normal individuals, the triplet repeat region is less than 50 triplets long (150 nucleotides). The identification of the characteristic expansion of the triplet repeat is diagnostic for the presence of the fragile X mutation. Individuals with expansions of 60-200 triplets are unaffected but may transmit the disorder (premutation); those with expansions of approximately 200 triplets may be affected and/or transmit the disorder; males with expansions of >200 triplets will be affected. The mothers of affected children are obligate carriers. Molecular genetic studies are indicated in other family members.

Reference: Sutherland GR et al. Med J Aust 1993; 158: 482-485.

HAEMOCHROMATOSIS

Specimen: 5-10mL blood in EDTA.

Method: PCR and restriction enzyme digestion or other method to detect single base changes in the HFE gene.

Application: Assists in the assessment of those with high ferritin levels. Diagnosis of haemochromatosis in patients with clinical or chemical evidence of the disease, or with a family history of haemochromatosis. Can be used in family studies to detect those who have the haemochromatosis mutation prior to the development of iron overload.

Interpretation: Approximately 1/300 northern Europeans have a genetic defect associated with haemochromatosis, although not all develop the clinical disease. About 85% of adult genetic haemochromatosis patients have the homozygous C282Y genotype, about 10% have the compound C282Y/H63D, and a few have the homozygous H63D genotype. C282Y is found in much lower frequencies in other populations eg Mediterranean or Asian. The significance of a second mutation (H63D) remains to be determined.

Reference: UK Haemochromatosis Consortium. Gut 1997; 41: 841-844.

HAEMOGLOBIN VARIANTS

Specimen: 5-20 mL blood in EDTA tube.

Method: PCR-based analysis.

Application: The diagnosis of haemoglobin variants, when appropriate after clinical assessment, FBC and haemoglobin electrophoresis. Prenatal diagnosis. See also Sickle Cell Disease below.

Interpretation: Characterisation of specific haemoglobin variants eg Hb E, Hb D, Hb Lepore. Prenatal diagnosis for at-risk pregnancies.

Reference: Weatherall DJ. Mol Med Today 1995; 1: 15-20.

HAEMOPHILIA

Specimen: 2-20 mL blood in EDTA tube.

Method: Southern blot or PCR-based analysis to detect common mutations in the factor VIII and factor IX genes.

Application: Used in conjunction with coagulation studies to define carrier status in female relatives of affected males. Prenatal diagnosis for haemophilia may be available if the mother carries an identified mutation.

Interpretation: The number and type of mutations tested varies between laboratories. The commonest mutation in Haemophilia A (factor VIII deficiency) is an inversion of intron 22 and this accounts for about one-third of factor VIII gene mutations in those who are severely affected.

Reference: Laboratory methods for the genetic diagnosis of bleeding disorders. Clin Lab Haem 1998; 20: 3-19.

HUNTINGTON DISEASE

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR analysis to detect an expanded triplet repeat mutation in the Huntington gene.

Application: Used to detect the Huntington disease mutation in symptomatic or asymptomatic people. Predictive tests in asymptomatic family members should only be arranged after consultation with the pathologist and a clinical genetics service. Prenatal diagnosis is available. The DNA test may also be used to assist in the differential diagnosis of a neurological problem which could be Huntington disease.

Interpretation: In the normal population, the triplet repeat region is less than 31 triplets in length whilst those with Huntington disease have repeats which are >37. The identification of the characteristic expansion of the triplet repeat is diagnostic for the presence of the Huntington disease mutation. Those with very high repeat numbers eg >50 repeats typically have early onset of disease. Individuals with triplet repeats between 31-37 are thought to have a premutation and although they may not develop Huntington disease, their offspring may be at risk. Care should be taken in interpreting the normal and pathological range for triplet repeats since there is some variability between laboratories. Similarly, the significance of the repeat numbers may be interpreted differently in some cases.

Reference: Rubinsztein DC et al. Am J Hum Genet 1996; 59: 16-22. ACMG/ASHG Statement: Laboratory guidelines for Huntington disease genetic testing. Am J Hum Genet 1998; 62: 1243-1247.

MITOCHONDRIAL DISORDERS

Specimen: Check with pathologist: blood is satisfactory for some disorders but liver or skeletal muscle biopsy may be required due to the variable involvement of mitochondria in different tissues (heteroplasmy).

Method: Southern blot or PCR-based analysis to detect selected point mutations, deletions or duplications in mitochondrial DNA.

Application: Used in conjunction with other studies in the diagnosis of mitochondrial disorders. These disorders are characterised by maternal transmission to all offspring but may also occur sporadically. Genetic studies may be used to identify presymptomatic relatives who carry the mitochondrial mutation. Prenatal diagnosis for mitochondrial disorders is usually not available.

Interpretation: The number and type of mutations tested varies between laboratories. The identification of a mitochondrial mutation does not predict the severity, age of onset, or tissue involvement of a mitochondrial disorder. Molecular genetic studies of other family members may be indicated. These methods do not detect all possible mutations and the lack of a defined mutation does not necessarily exclude the clinical diagnosis.

Reference: Byrne E. Med J Aust 1991; 154: 646-647.

MULTIPLE ENDOCRINE NEOPLASIA TYPE 2 (MEN2)

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analysis to detect mutations within the ret gene.

Application: Used in conjunction with endocrine studies to diagnose MEN2 and to identify affected family members prior to clinical presentation.

Interpretation: Current laboratory methods detect over 95% of the mutations in the ret gene. The identification of one of the characteristic mutations within the ret gene is diagnostic for MEN2, but does not predict the age of onset. Molecular genetic studies may be indicated in other family members.

Reference: Lips CJ et al. N Engl J Med 1994; 331: 828-835.

MYOTONIC DYSTROPHY

Specimen: 10-20 mL blood in EDTA tube.

Method: Southern blot or PCR analysis to detect an expanded triplet repeat mutation in the myotonin protein kinase gene.

Application: Used to detect the myotonic dystrophy mutation in symptomatic or asymptomatic people. Prenatal diagnosis for myotonic dystrophy is available if the mother has been shown to have an abnormal gene.

Interpretation: In the normal population, the triplet repeat region is less than 30 triplets (90 nucleotides) in length. More than 99% of patients with typical myotonic dystrophy have more than 50 triplets in this region. The identification of the characteristic expansion of the triplet repeat is diagnostic for the presence of the myotonic dystrophy mutation. The degree of expansion of the triplet region is correlated with the age of onset and severity of the disorder, but considerable variation exists. Molecular genetic studies may be indicated in family members.

Reference: Shelbourne P et al. N Engl J Med 1993; 328: 471-475.

PROTHROMBIN MUTATION

Specimen: 5-10 mL blood in EDTA tube.

Method: PCR-based analysis.

Application: Aids in the investigation of thrombophilia of possible congenital origin.

Interpretation: Prothrombin gene mutation (prothrombin G-->A20210 ) was first reported in 1996 to be associated with venous thrombosis. In a population-based case-control study, the 20210 A allele was identified as a common allele (allele frequency, 1.2%; 95% confidence interval, 0.5% to 1.8%), which increased the risk of venous thrombosis almost threefold. The risk of thrombosis increased for all ages and both sexes. An association was found between the presence of the 20210 A allele and elevated prothrombin levels. Elevated prothrombin itself also was found to be a risk factor for venous thrombosis. The increased prevalence of this allele was independent of the presence of the factor V Leiden mutation. In one study looking at unselected patients with deep venous thrombosis, 34% (one patient carried both traits) were carriers of either mutation.

This prothrombin mutation has also been linked with cerebral venous thrombosis (especially in women and, in particular, those who are current users of oral contraceptives) and myocardial infarction, especially in the presence of other risk factors.

References: Poort SR et al. Blood 1996; 88: 3698-3703. Hillarp A et al. Thromb Haemost 1997; 78: 990-992. Martinelli I et al. N Engl J Med 1998; 338: 1793-1797. Doggen CJ et al. Circulation 1998; 97: 1037-1041.

SICKLE CELL DISEASE

Specimen: 5-10 mL blood in EDTA tube.

Method: PCR-based analysis to detect the sickle mutation in the b globin gene.

Application: Prenatal diagnosis is available if both parents are heterozygous for the sickle cell gene (sickle cell trait).

Interpretation: Homozygous and heterozygous sickle cell anaemia are diagnosed by FBC and tests for the presence of haemoglobin S (incl haemoglobin electrophoresis); molecular genetic studies may permit prenatal diagnosis. A compound heterozygote for beta thalassaemia and HbS trait can have a similar clinical picture to sickle cell disease. Detection of the latter may only be possible following a family study.

Reference: BCSH General Haematology Task Force. J Clin Pathol 1994; 47: 199-204.

SPINAL MUSCULAR ATROPHY

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analysis to detect the presence of a deletion in the SMN (survival motor neurone) gene.

Application: Used in conjunction with muscle biopsy in the diagnosis of severe or intermediate spinal muscular atrophy. Not useful for identifying carriers. Limited application in evaluation of older children or adults with recessive spinal muscular atrophy; not indicated in autosomal dominant spinal muscular atrophy. Prenatal diagnosis for severe and intermediate forms of spinal muscular atrophy is available.

Interpretation: Approximately 90% of children with severe or intermediate forms of spinal muscular atrophy have deletions in both copies of this gene. The identification of a deletion in both copies of the SMN gene in a symptomatic person is diagnostic of spinal muscular atrophy. These methods do not detect all possible mutations. The lack of deletions does not necessarily exclude the diagnosis, and family studies may allow prenatal diagnosis.

Reference: Lewin B. Cell 1995; 80: 1-5.

SPINO-BULBAR MUSCULAR ATROPHY

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analysis to detect an expanded triplet repeat mutation in the androgen receptor (AR) gene.

Application: Used for definitive diagnosis in symptomatic males. Used to define carrier status in female relatives of affected males. Predictive tests in asymptomatic males should only be arranged in consultation with the pathologist and a clinical genetics service. Prenatal diagnosis may be available if the mother carries an identified mutation.

Interpretation: In the normal population, the triplet repeat region in the AR gene is less than 40 triplets long (120 nucleotides). An expanded triplet repeat accounts for almost all cases of spino-bulbar muscular atrophy. The identification of a mutation is diagnostic of spino-bulbar muscular atrophy (symptomatic male) or of carrier status (asymptomatic female). The identification of this mutation does not predict the severity or age of onset of the disorder. This test does not distinguish between asymptomatic and affected individuals. Molecular genetic studies may be indicated in other family members.

Reference: Muller U et al. J Neurol Sci 1994; 124: 119-140. La Spada AR et al. Ann Neurol 1994; 36: 814-822.

SPINO-CEREBELLAR ATAXIAS (SCA)

Specimen: 10-20 mL blood in EDTA tube.

Method: PCR-based analyses to detect expanded triplet repeat mutations known to cause various autosomal dominant spinocerebellar ataxias (eg SCA1, Machado-Joseph disease).

Application: Used to detect a triplet repeat mutation in symptomatic or asymptomatic individuals. Predictive tests in asymptomatic family members should only be arranged after consultation with the pathologist and a clinical genetics service. Prenatal diagnosis is available.

Interpretation: Expanded triplet repeats in one of the genes account for approximately half of the cases of autosomal dominant SCA. The identification of the characteristic expansion of the triplet repeat is diagnostic for the presence of a mutation that can cause the particular SCA. There is some correlation between the specific mutation and the age of onset and severity of the disorder, but considerable variation exists. Molecular genetic studies may be indicated in other family members.

Reference: Muller U et al. J Neurol Sci 1994; 124: 119-140. La Spada AR et al. Ann Neurol 1994; 36: 814-822.

THALASSAEMIA

Specimen: 5-20 mL blood in EDTA tube.

Method: Southern blot or PCR-based analysis to detect mutations in the a globin or b globin genes.

Application: Prenatal diagnosis is available if both parents have heterozygous b thalassaemia or the type of a thalassaemia (alpha-zero thalassaemia) which will give rise to Hb Bart’s hydrops fetalis..

Interpretation: The number and type of mutations tested varies between laboratories, but, in general, 70-90% of all mutations responsible for thalassaemia should be detectable.

Reference: Trent RJ et al. Prenatal diagnosis for thalassaemia in a multicultural society. Prenatal Diagnosis 1998; 18: 591-598.

---