Background

Achondroplasia is inherited as an autosomal dominant with complete penetrance, although the majority (80-90%) of cases are sporadic. Estimated disease frequency is between 1/15,000 and 1/40,000. Clinically achondroplasia is characterized by macrocephaly, craniofacial disproportion, midface hypoplasia and dysplasia of the metaphysis of the tubular bone. Short limbs with a normal trunk are remarkably consistent. Achondroplasia exhibits considerable genetic homogeneity, because specific mutations correlate strongly with the disease.

99% of Achondroplasia has been attributed to the G380R mutation. Most cases of this mutation were due to a G to A transition (98%), and the remainder to a G to C transversion (1%) at nucleotide 1138 in the transmembrane domain of the FGFR3 gene

Indication for testing

Diagnostic testing for individuals affected by Achondroplasia

Prenatal testing can also be offered.

Genetic testing

DNA testing for the G380R mutation

Background

The BRAF protein is a member of the serine-threonine kinase RAF family and plays a role in the RAS-RAF-MEK-ERK pathway leading to enhanced survival of certain cells. BRAF mutant tumors represent a discrete subset of colorectal cancer (CRC) characterized by poor overall survival, unique patterns of metastatic spread, and limited response to current chemotherapy and targeted therapies. The BRAF V600E mutation at position 1799 on exon 15 occur in about half of melanomas and in 10-15% of colon cancers.

The BRAF V600E mutation is associated with resistance to antibody therapy against the EGF receptor. The mutation status is evaluated to:

• identify cutaneous or non-cutaneous melanomas that may benefit from anti-RAF treatment (drugs that target BRAF)
• rule out Lynch syndrome (HNPCC) in patients with microsatellite unstable cancer
• differentiate classical HCL (Hairy Cell leukemia) from HCL-like disorders.

Indication for testing

Patients with colorectal cancer or melanoma.

Genetic testing

DNA analysis of the V600E BRAF mutation.

Background

Calreticulin (CALR) mutations have been found to be present in 67-88% of JAK2 and the thrombopoietin receptor gene (MPL) unmutated essential thrombocythaemia (ET) and primary myelofibrosis (PMF) cases. CALR mutations may be present in a very low frequency in other myeloid malignancie (MDS, atypical CML or CMML). A positive result may not distinguish between ET and PMF or other myeloid neoplasms. Very rarely, CALR mutations have been found with a concurrent JAK2 mutation.

Indication for testing

Diagnosis of myeloproliferative neoplasms (MPN), in particular essential thrombocythaemia (ET) and primary myelofibrosis (PMF) in JAK2 negative cases.

Genetic testing

Screening for somatic insertion/deletion-type mutations in exon 9 of the CALR gene.

Background

Chimerism testing (engraftment analysis) is performed for patients who have received a hematopoietic stem cell transplant. The test involves identifying the genetic profiles of the recipient and of the donor and then evaluating the extent of mixture in the recipient’s blood or bone marrow after the transplant. DNA is isolated from the recipient and potential donor before the transplant and analysed to determine whether the genetic markers unique to the donor and the recipient are sufficient to distinguish the donor from the recipient. After the transplant takes place, the performance of the transplant engraftment is assessed by evaluating the donor versus recipient. When a mixed profile occurs in the post-transplant sample of the recipient, the peak area from the informative alleles are used to calculate the percentage of the recipient DNA present in the sample. This data is reported together with successive historical data to monitor the patient’s progress.

Indication for testing

Individuals about to receive a bone marrow transplant or patients who had received a transplant and are being monitored as well as their donor’s.

Genetic testing

A subset of 21 STR markers are used for profile establishment of the recipient and donor before and after transplant and this can be used to monitor the recipient’s progress.

Background

Familial hypercholesterolemia (FH) is characterized by elevated cholesterol levels, specifically very high low-density-lipoprotein (LDL) levels in the blood and early cardiovascular disease. The LDL receptor protein normally removes LDL from the circulation. FH is an autosomal dominant disorder, which occurs in 1:500 people in most countries, but in 1:80 in the Afrikaner population in SA. Homozygous FH is much rarer, occurring in 1 in a million births and causes accelerated atherosclerosis in childhood. FH is caused mainly by mutations in the low-density lipoprotein receptor (LDLR) gene. Three founder mutations account for ~90% of FH cases among the Afrikaners in SA.

Familial defective apolipoprotein B100 (FDB) is due to a mutation in the apolipoprotein B (APOB) gene, that markedly reduces the affinity of the LDL receptor. FDB is characterised by moderately raised to high plasma cholesterol levels.

Indication for testing

• Diagnostic testing of individuals affected with FH
• Elevated levels of total cholesterol
• Tendon xanthomata (TX) in patient
• Family history of elevated cholesterol and heart disease
• Family history of total cholesterol >7.5mmol/l

Genetic testing

• Screening for three LDLR founder mutations, D227E, V429M and D175N.
• Screening for the R3500Q mutation in the APOB gene is also performed.

Background

Fanconi anaemia (FA) is a chromosome instability syndrome with progressive bone marrow failure and an increased risk for cancers. Common manifestations include growth retardation, skin abnormalities (hyperpigmentation and café-au-lait spots), radial axis defects and less frequently, renal anomalies, hypogonadism, mental impairment, heart defects and diabetes mellitus. FA is seen most often in children, with a mean age of onset of 8 years. There is a 15,000-fold increased risk of MDS and AML in FA patients, with a mean age of developing a haematological malignancy at 13-15 years of age 2. FA is an autosomal recessive disorder and has a worldwide incidence of 1 in 300 000. South African Afrikaner, Black and Jewish FA patients have been shown to carry founder mutations in the FANCA (79%), FANCG (80%) and FANCC, respectively.

Indication for testing

• Diagnostic testing for FA
• Carrier and prenatal testing for individuals with a family history of FA

Genetic testing

• FANCA screening for large deletions and 3398delA
• FANCG screening for the 7-bp deletion, c.637_643delTACCGCC
• FANCC screening for the splice site mutation, IVS4+4A>T

Background

Acute Myeloid Leukemia (AML) in general has a poor prognosis. FLT3 (fms-like tyrosine kinase 3) is a receptor tyrosine kinase that is normally expressed on many cell types including hematologic stem cells. Assessment of the mutation status of the FLT3 receptor gene in karyotype normal AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presence of FLT3 activating mutations portends a poor prognosis. For this reason FLT3 activation mutation testing is required to stratify disease and determine appropriate treatment options. This PCR assay targets regions of the FLT3 gene to identify internal tandem duplication (ITD) mutations and tyrosine kinase domain (TKD) mutations, such as the D835. Mutation of the FLT3 receptor, either by internal tandem duplication (ITD) of the juxtamembrane domain or by point mutation of the aspartic acid residue D835 in the activation loop of the kinase domain, causes constitutive activation of the FLT3 receptor.

FLT3 Internal Tandem Duplication (ITD)/length mutations are caused by duplication and insertion of a portion of the FLT3 gene that includes the region in and around the juxtamembrane (JM) region of the FLT3 gene. These mutations vary in both the location and the length of the inserted duplicated DNA sequence. ITD mutations result in constitutive autophosphorylation and activation of FLT3.

FLT3 Tyrosine Kinase Domain (TKD) mutations are caused by nucleic acid substitutions that result in a change in the amino acid sequence in this highly conserved catalytic center. TKD mutations, such as D835, result in constitutive autophosphorylation and activation of FLT3.

Mutations in nucleophosmin (NPM1) which almost always involve a 4-bp insertion (that varies) in a limited region of exon 12, causes aberrant cytoplasmic expression of nucleophosmin (NPMc+). A NPM1 mutation leads to a more favorouble prognosis in patients with AML which have a normal karyotype.

The clinical impact of NPM1 mutations is affected by the mutational status of the FLT3 gene. It has been proposed that combining the status of these two mutations allows for stratification into 3 prognostic groups: (1) Good prognosis FLT3-/NPM1+, (2) Intermediate prognosis FLT3-/NPM1- or FLT3+/NPM1+, (3) Poor prognosis FLT3+/NPM1-.

Indication for testing

• Determine prognosis in patients with AML with a normal karyotype
• Risk stratification and patient treatment

Genetic testing

• DNA analysis of FLT3 ITD and D835 mutations and a 4bp insertion in exon 12 of NPM1.

Background

Fragile X syndrome, or Martin-Bell syndrome, is a syndrome of X-linked mental retardation. Boys with the syndrome may have large testicles (macroorchidism), prognathism, hypotonia and autism, and a characteristic but variable face with large ears, long face, high-arched palate, gynecomastia, and malocclusion. Additional abnormalities may include lordosis, heart defect, pectus excavatum, flat feet, shortening of the tubular bones of the hands, and joint laxity. Females who have one fragile chromosome and one normal X chromosome may range from normal to mild manifestations of fragile X syndrome.

The syndrome is associated with the expansion of a single trinucleotide gene sequence (CGG) in the promoter region of the FMR1 gene on the X chromosome (Xq27.3), and results in a failure to express the FMR1 protein which is required for normal neural development. Expansion of the CGG repeating codon to such a degree results in a methylation of that portion of the DNA, effectively silencing the expression of the FMR1 protein. Mutation of the FMR1 gene leads to the transcriptional silencing of the fragile X-mental retardation protein, FMRP. In normal individuals, FMRP is believed to regulate a substantial population of mRNA. FMRP plays important roles in learning and memory, and also appears to be involved in development of axons, formation of synapses, and the wiring and development of neural circuits.

Indication for testing

• Individuals suspected to be affected with Fragile X syndrome.
• Carrier and prenatal testing of individuals with a family history of Fragile X syndrome.

Genetic testing

• DNA analysis to determine the FMR1 CGG repeat length.
• This assay is not able to detect point mutations or deletions within the FMR1 gene and provide no information on the methylation status of the FMR1 promoter.

Background

The myeloproliferative disorders (MPD) are a group of haematological conditions where there is a primary disorder at the level of the multi-potent haematopoietic stem cells leading to increased production in one or more blood cell types.

Leukocytes from the majority of polycythemia vera (PV) patients and up to half of essential thrombocythemia (ET) patients and idiopathic myelofibrosis (IMF) patients have been found to carry the activation V617F mutation in the auto-inhibitory domain of the Janus Kinase 2 gene.

The V617F mutation also has been found in a minority of patients with other myeloid stem cell disorders, including chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS).

The common JAK2 mutation found is a single nucleotide substitution G>T at position 1849 resulting in a Valine to Phenylalanine substitution at codon 617 (V617F). This mutation renders the kinase constitutively active independent of ligand binding.

Identification of the JAK2 (V617F) mutation in myeloproliferative disorders (MPD) may be useful both to assist in identifying MPD and assessing patients for a viable targeted pharmacological treatment.

Indication for testing

Patients suspected to have PV, ET or IMF as well as CMML and MDS.

Genetic testing

• DNA analysis for the V617F JAK2 mutation.

Background

MPL is involved in megakaryocyte development and platelet production as well as hematopoietic stem cell homeostasis by encoding the thrombopoietin receptor which binds to thrombopoietin.

MPL mutations have been found to be present in 5-10% of JAK2/CALR unmutated ET and PMF cases.

MPL mutations may be present in a very low frequency in other myeloid malignancies (MDS, atypical CML or CMML). A positive result may not distinguish between ET and PMF or other myeloid neoplasms.

Indication for testing

Detection of MPL mutations is used for diagnosis and monitoring of patients with myeloproliferative neoplasms (MPN) and suggest either essential thrombocythemia (ET) or primary myelofibrosis (PMF) in a subset of cases in which JAK2 testing is negative.

Genetic testing

• Mutation analysis testing for the W515L (TGG>TTG) and W515K (TGG>AAG) mutations in exon 10 of the MPL gene.

Background

Lymphoplasmacytic lymphoma (LPL) is a distinct B-cell lymphoproliferative disorder primarlily characterized by bone marrow infiltration of lymphoplasmacytic cells. When LPL produces a serum monoclonal immunoglobulin of IgM class, it may be classified as Waldenstorms macroglobulinemia (WM). Based on the pathologic and phenotypic features of the tumour, it is difficult to differentiate between WM and similar B-cell tumors that may also have plasmacytic differentiation and/or secretion of IgM. Somatic mutations of MYD88 which is a key component of the Toll-like receptor signaling machinery is detected in about 90% of LPL. The somatic mutation may be observed less frequently in other small B-cell lymphomas such as marginal zone lymphoma (MZL), including nodal, splenic, or extranodal mucosa-associated lymphoid tissue (MALT). A somatic variant (T to C) at position 794 in MYD88 gene that results in an amino acid change from leucine to proline (L265P) to help distinguish this low-grade B-cell lymphoma from other B-cell lymphoma types.

Indication for testing

Distinguishing lymphoplasmacytic lymphoma from other low-grade B-cell lymphoproliferative disorders which may be in the differential diagnosis.

Genetic testing

• DNA analysis of the L265P mutation.

Background

Prader-Willi syndrome (PWS) is a complex genetic condition that affects many parts of the body. In infancy, this condition is characterized by weak muscle tone (hypotonia), feeding difficulties, poor growth, and delayed development. Beginning in childhood, affected individuals develop an insatiable appetite, which leads to chronic overeating (hyperphagia) and obesity. Some people with Prader-Willi syndrome, particularly those with obesity, also develop type 2 diabetes. People with Prader-Willi syndrome typically have mild to moderate intellectual impairment and learning disabilities as well as behavioral problems.

Angelman syndrome (AS) is also a complex genetic disorder that primarily affects the nervous system. Characteristic features of this condition include delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia).

PWS and AS are distinct neurogenetic disorders, however both usually caused by chromosomal deletions on chromosome 15q11 or by uniparental disomy (UPD). Normal individuals have one copy of the unmethylated paternal allele and one copy of the methylated maternal allele. Two methylated alleles from a maternal disomy will appear in the case of PWS or two unmethylated alleles from a paternal disomy will indicate AS.

Indication for testing

• Diagnostic test for AS or PWS.

Genetic testing

• Microdeletions in the AS/PWS critical region as well as methylation status of chromosome 15q11.

Background

Molecular techniques have been developed as a rapid prenatal diagnostic tool to aid clinicians in diagnosing the most common chromosome disorders (trisomies 21, 13, 18 and sex chromosome aneuploidies) within 3 working days. Commercial multiplex kits using the Quantitative Flourescent Polymerase Chain Reaction (QF-PCR) method are available and used on DNA from cells of fetal origin. Using PCR amplification, fluorescent dye labeled primers target highly polymorphic regions of DNA sequences, short tandem repeats (STRs) that are located on the chromosomes of interest. Autosomal markers are used to detect trisomies of chromosomes 13, 18 and 21. Additional markers on the sex chromosomes (X and Y) can detect the most common sex chromosome aneuploidies. The QF-PCR test is used as a preamble to full chromosome analysis but has been offered as a stand-alone test to women who are at a relatively lower risk for a chromosome abnormality in their current pregnancy. However, the test is specific and only the chromosomes in question will be investigated. This may mean that if an abnormality is present in any of the other autosomes, it will not be detected.

Indication for testing

• Advanced maternal age.
• Increased risk identified by maternal serum screening programs.
• Fetal ultrasonography indicating increased risk.
• Previous child with a chromosomal abnormality.

Genetic testing

• Multiplex QF-PCR for specific STR’s on chromosomes 13, 18, 21 and X and Y.

Background

Molecular techniques have been developed as a rapid diagnostic tool to aid clinicians in diagnosing the most common chromosomal disorders involved in miscarriages (trisomies 13, 15, 16, 18, 21, 22 and sex chromosome aneuploidies as well as triploidy). Commercial multiplex kits using the Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) methods are available and used on DNA from cells of fetal origin. Using PCR amplification, fluorescent dye labeled primers target highly polymorphic regions of DNA sequences, short tandem repeats (STRs) that are located on the chromosomes of interest. Autosomal markers are used to detect trisomies of chromosomes 13, 15, 16, 18, 21 and 22 as well as triploidy. Additional markers on the sex chromosomes (X and Y) can detect the most common sex chromosome aneuploidies. The QF-PCR test is used to replace full chromosome analysis as cell culture is often unsuccessful on products of conception. However, the test is specific and only the chromosomes in question will be investigated. This may mean that if an abnormality is present in any of the other autosomes, it will not be detected.

Indication for testing

• Recurrent miscarriages
• Previous child with a chromosomal abnormality
• Any other abnormalities seen on ultrasound

Genetic testing

• Multiplex QF-PCR for specific STR’s on chromosomes 13, 15, 16, 18, 21, 21 and X and Y.
• If the QF-PCR is unsuccessful or uninformative FISH testing can also be conducted to detect aneuploidies of chromosomes 13, 18, 21 and the sex chromosomes.

Background

Variegate porphyria (VP) is an autosomal dominant inherited disorder of haem metabolism. VP is associated with decreased protoporphyrinogen oxidase activity (PPO). The disorder is characterized by photosensitivity and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia.

The activity of this enzyme is reduced by 50 percent in most people with VP. In severe cases that begin early in life, the enzyme is almost completely inactive. Nongenetic factors such as certain drugs, stress, and others listed above can increase the demand for haem and the enzymes required to make haem. The combination of this increased demand and reduced activity of protoporphyrinogen oxidase disrupts haem production and allows byproducts of the process to accumulate in the liver, triggering an acute attack. The R59W mutation in the PPO gene is associated with VP and is seen as a founder mutation in Africa.

Indication for testing

• Diagnostic testing for individuals affected by VP.
• Testing of family members affected with VP.

Genetic testing

• DNA analysis of the R59W mutation.

Background

Microdeletions in the Y chromosome have been found at a much higher rate in infertile men than in fertile men. Many of the deletions have been found in the azoospermia factor locus (AZF), at Yq11. Deletions in the AZFa, AZFb and AZFc/DAZ regions of the Y-chromosome occur in men with non-obstructive azoospermia and severe oligozoospermia. Sperm retrieval is ineffective for males with SCOS (Sertoli-cell-only syndrome) associated with AZFa and AZFb deletions, but has been achieved for males with AZFc deletions.

Indication for testing

• Male infertility

Genetic testing

DNA testing is performed for key functional regions associated with Azoospermia Factor (AZF), namely the regions that flank AZFa and cover AZFb, AZFc, AZFd including DAZ, KAL-Y, SMCY and flanking loci for other key spermatogenesis-related genes (namely RBM1, DFFRY and DBY).