SELECTED STUDIES SUPPLEMENT
MOLECULAR DIAGNOSIS AND GENOTYPE-PHENOTYPE CORRELATION: A USEFUL TOOL IN PUBLIC HEALTH
By Prof. Habiba Bouhamed-Chaabouni
Modern molecular medicine encompasses the utilization of many molecular biological techniques in the analysis of disease, disease genes and disease gene function. The number of genetic disorders that can be diagnosed is constantly expanding. Molecular diagnosis of genetic disorders is today widespread medical facility through the world.
During the last part of the twentieth century, advances in molecular biology and cell biology have provided us with an understanding of the mechanisms of disease at the molecular level. This understanding can now be translated into diagnostic, prognostic, and therapeutic tools. It is now possible to phenotype and genotype human tumours, to increase the accuracy and reproducibility of pathologic diagnosis. Abnormal molecules not only provide a signature for the presence of a disease, but may also provide the indication for a drug targeting the specific abnormal function. This circumstance creates a particularly tight link between diagnostics and therapeutics and has a good impact on public health.
Molecular Tools For in situ gene investigation
Thanks to molecular probes we can realize in situ genome investigation, directly on tissue samples and cells. Fluorescent in situ hybridization (FISH) is available for analyzing the gene or the chromosome region on chromosomes, nucleus, and cells.
Molecular Tools are used for gene investigation after DNA or RNA extraction. Several nucleic acid analysis techniques are available and routinely used such as:
§ Restriction Fragment Polymorphism
§ The Polymerase Chain Reaction and related techniques
§ DNA Sequencing
§ Cloning of complimentary DNA (cDNA)
§ The Ligase Chain Reaction, LCR
§ Microarray Analysis and many others
Molecular Tools are useful in medical practice for Diagnosis, Genotype-Phenotype correlation, Prognosis and Screening. Contributions of molecular testing have been particularly dramatic for the diagnosis of several disorders and may be the unique way to confirm diagnosis.
DIAGNOSIS
How molecular analysis reduces patient invasive investigation; the example of Duchenne Muscular Dystrophy (DMD)
DMD is an X-linked recessive muscular dystrophy which affects boys. After normal childhood the walk regression appears at age 3 to 4 years with muscle hypertrophy. The natural course of the disease leads to progressive muscle loss function.
The disease is related to muscle fibbers degeneration with lack of “dystrophin” in muscular cells.
Few years ago, the diagnosis was based on
- Clinical features
- Electromyogramme, and
- Muscle biopsy which shows absence of Dystrophin as indicated on figures 1,2.
Differential diagnosis with similar muscular dystrophies was based only on muscle biopsy analysis looking for presence or absence of the specific protein, the dystrophin.
In 1986 the Dystrophin gene was localised on X p 21.2 and then identified.
Gene analysis showed the presence of large gene deletion causing the DMD, while small gene deletion is causative of Becker muscular dystrophy (BMD), a less severe clinical expression.
Gene deletion is present in about 70% DMD patients, and confirms diagnosis. Elsewhere point mutations are present. Today invasive investigation as muscle biopsy is replaced by molecular analysis of the Dystrophin gene.
GENOTYPE-PHENOTYPE CORRELATION: the Example of Congenital Adrenal Hyperplasia.
CAH is an autosomal recessive disorder due to defective enzyme in biosynthesis chain of cortisol in adrenal gland. The most common one is related to 21-hydroxylase deficiency resulting from molecular defect in the steroid 21-hydroxylase (CYP21) gene.
The phenotype corresponding to homozygous deleterious genes is variable. First the external genitalia development is sex influenced, since all affected males have normal external genitalia while females may present ambiguous genitalia. Second we distinguish three main clinical expressions.
In the classic salt-wasting (SW) form, the most severe form, patients suffer from renal salt loss due to the lack of aldosterone as well as virilization due to accumulated adrenal androgen. In the classic simple virilizing (SV) form, patients also undergo virilization. In the nonclassic form, patients lack the neonatal symptoms and present with late-onset androgen excess, such as pseudoprecocious puberty and hirsutism. These phenotypes are caused by different mutations of CYP21 gene. Genetic lesions causing 21-OH deficiency are large lesions, including deletions and conversion with the CYP21P, a pseudo-gene situated in tandem with the functional gene (fig.3). large lesions represent 25% of 21-OHD alleles, and point mutations correspond to 75% of 21-OHD alleles. Several mutations in CYP21 gene have been described.
Generally the severity of the phenotype depends on mutations present on both alleles. Large deletions, Q318X and R356W mutations found in our series, were always associated with the SW form, whereas the IVS2–13A/C3G mutation was found in either SW or SV, and I172N mutation was associated with SV. V281L mutation is less severe and is present in non-classic forms (fig.4).
Phenotype-genotype correlation is of a great help for patients medical care and for genetic disorders prevention.
Molecular Analysis Strategy.
Molecular epidemiological analysis of CYP21 defined the common mutations among Tunisian population, the commonest one was Q318X. Regarding these results, we established a strategy for CYP21 alleles screening for affected patients and their families. The strategy is following:
We realize specific amplification of CYP21 using specific primers. The absence of gene amplification means that there is large lesion, so we screen large deletion and conversion. In case of amplification, restriction enzyme digestion of PCR-amplified DNA is used to detect the presence of six more common point mutations: Q318X, then P30l; IVS2-13 A/C ®G; I 172N; CL6; V281L
Finally if neither deletion nor mutations are found we go for gene sequencing. This methodology is used both for postnatal and prenatal diagnosis which never leads to pregnancy termination in this pathology.
PHENOTYPE-GENOTYPE CORRELATION AND PROGNOSIS
Phenotype-genotype correlation is available for prognosis appreciation for some genetic diseases. Molecular analysis let us predict the phenotype in familial Mediterranean fever (FMF) for example.
Familial Mediterranean Fever (FMF) is one of the periodic fever syndromes, and is extremely common among Arabs. It is an autosomal recessive disorder characterized by recurrent, short, self limited , and painful episodes of fever and polyserositis. The gene responsible for FMF, MEFV, located on chromosome 16p13.3, comprises 10 exons and 781 codons (fig.5). The product of MEFV gene, named pyrin/Marenostrin is expressed in polymorphonuclear cells and monocytes and it is proposed that it regulates inflammatory responses at the level of leukocyte cytoskeleton organization.
The treatment is Colchicine drug and if precociously prescribed it can prevent complications.
Clinical diagnosis of the disease is unclear in several cases since clinical features are not specific. Colchicine test may help the physician for treatment decision but diagnosis confirmation is available only by the presence of MEFV gene mutations. Identification of MEFV mutations is useful to determine the phenotype since there is a good genotype–phenotype correlation especially for amyloidosis and renal failure complication. Mutation M694V (exon10) has severe expression and affected patients are at risk of amyloidosis while E148Q (exon2) mutation has moderate to normal expression. M680I (exon10) has severe expression but low rate of amyloidosis. Genotype analysis confirms diagnosis, predict prognosis which let physician adapt medication.
GENETIC HETEROGENEITY
Genetic heterogeneity makes genetic counselling sometimes very difficult. Gene determination and family members’ genotype analysis are needed to give pertinent counselling. Bardet-Biedl Syndrome (BBS) is a good illustrative example. Described by Biedl (1922) and Bardet (1920) , BBS is today a well defined syndrome. The common clinical features are obesity, pigmentary retinopathy, postaxial polydactyly, hypogonadism, renal disorders and highly variable mental retardation. It is an autosomal recessive disease due to several genes listed up to date to 10 different loci, most with identified genes. Molecular identification is necessary for genetic counselling and prenatal diagnosis.
MOLECULAR TOOLS USED IN CYTOGENETICS
Molecular methods are used to infra-microscopic chromosomal anomalies determination. Several microdeletion syndromes have been discovered with the FISH method, such as the Prader–Willi and Angelman syndromes, the DiGeorge and velocardiofacial syndromes. These disorders seem to represent the consequence of the simultaneous deletion of a group of genes, rather than the mutation of a single gene. In some cases, definition of chromosome rearrangement leads to well define the phenotype and to propose candidate genes for clinical features.
There are so many other applications of molecular analysis for screening, genetic testing in different genomics fields. In cancer the possibility of determining an individual's susceptibility to cancer at birth, and at selected periods during his or her life is capital. Other domains like cardiogenomics, behavior genomics and complex diseases are at the origin of biotechnological evolution.
Today with microarrays and chips where more than one hundred genes are spotted we can analyse in one time several genes and mutations.
Molecular analysis is available for gene anomalies, for chromosomal micro-deletions genetic disorders, for cancer, for micro-organisms detection and non genetic disorders. Using these techniques gives us large information about individual, family, population and ethnic groups. Respect of ethical conditions is mandatory in case.
In practice, the principal limitation in molecular diagnosis is the heterogeneity of genetic changes that underlie inherited disorders. A wide variety of changes, ranging from complete deletion of a gene to substitution of one nucleotide base in the DNA for another, can disrupt genetic function. Families studies are necessary to determine the gene modifications and to define whom members are or not at risk. Molecular tools for gene investigation are applicable in different tissues and at different stages of organism life: blastula embryo, fœtus, post-natal, post-mortem periods.
Molecular diagnosis is helpful for prevention of severe disorders, which makes genetic counselling easy and efficient. Prenatal diagnosis and preimplantation diagnosis will be a part of prevention.
CONCLUSION
With development of molecular tools we will promote deep knowledge of genes activity and interactions, have adapted tools for diagnosis for better prevention of genetic disorders including complex diseases and we will be able to adapt treatment and drugs.
Fig3: CYP21 gene situation on
the short arm of chromosome
Fig4:
Common mutations among 21-hydroxylase
deficiency alleles: CYP21 gene mutations. Red square:
severe phenotype, yellow square : mild phenotype, green square :moderate
phenotype.
Professor Habiba Bouhamed-Chaabouni is an international medical geneticist working at the Department of congenital and hereditary disorders, Charles Nicolle hospital & Medical School.Tunis, Tunisia. This study was presentedat the First International Kuwait Medical Genetics Conference in March 2006 .
