Finding Genes For Mendelian Disorders Biology Essay


1. Introduction

1.1 Mendelian disorder

Mendelian disorders or single-gene disorders are caused by a mutation in a single gene which high penetrance and decided by if it makes protein function abnormal or not. It follows Mendel's law of inheritance. Online Mendelian Inheritance in Man at shows that there are nearly 20,000 diseases caused by single-gene mutations up to now. There are commonly five modes of inheritances-autosomal dominant inheritance, autosomal recessive inheritance, x-linked autosomal inheritance, x-linked recessive inheritance and mitochondrial inheritance. Moreover autosomal dominant inheritance can have three categories: complete dominance inheritance, incomplete dominance inheritance and co-dominance inheritance, (irregular dominance inheritance, (delayed dominance inheritance, sex-influenced dominance inheritance

1.2 Isolated populations

Venken and Del-Favero (2007, 1157) suggest that due to increased inbreeding and genetic drift in isolates, certain alleles will be present more frequently in the population, while others are lost increasing genetic homogeneity. Moreover, isolated populations did not experience a large degree of admixture with surrounding populations for generations due to geographic, cultural and religious barriers, so they have small gene pool. As a result, isolated populations reduce genetic complexity and minimize environmental component of disease.

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The most common and powerful approach for identifying a disease genes for mendelian disease is positional cloning. There can be three steps for the whole procedure: defining minimal candidate region and identifying candidate gene within it and mutation screening. It is not compulsory to identify the chromosomal location for candidate genes, but it is common to define the candidate chromosomal region before identifying candidate genes. It is most important to minimize the candidate region when defining candidate region so that the number of genes are not very large. Furthermore, enough pedigrees should be obtained to gain satisfactory and strong enough results.

2 Defining candidate region

2.1 Fining-Mapping

Polymorphic markers are essential for genetic linkage analysis. If a family member inherits disease from parents he/she also receives markers lie near the disease gene allele as at least one of markers lie close enough to the disease gene. Genetic mapping requires double heterozygote, which requires different alleles at both loci. People heterozygous for two different diseases are extremely rare. For this reason, human genetic mapping depends on markers.

Marker locus must be highly polymorphic which so that there is a reasonably good chance of a person with a disease gene of interest also being heterozygous at the marker locus and then it is useful for linkage analysis. Markers help scoring genetic variation easily and cheaply once the markers are sufficiently polymorphic. The interval of Markers should not be greater than 10-20cM. Strachan & Read (2003, 402) say that we need at least 300 markers for imperfect informativeness and usually 350 markers are enough.

In conclusion, linked markers should be co-dominant which help us determine the phases easier, numerous and highly polymorphic. High degree of polymorphism increase the probability that matings will be informative. Two marker types are commonly used in genetic mapping: microsatellites and SNPs.

2.1.1 Microsatellites

Turnpenny and Ellard(2007, 69) discuss in their book that Microsatellites also known as Short Tandem Repeat Polymorphism is the repeats of CA/GT. They are highly polymorphic because the different number of repeats at one site among people. These polymorphic repeats can be detected by using PCR to amplify small segments of DNA containing the tandemely repeated units, followed by electrophoresis in a gel suitable for resolving single nucleotide differences in size.

From figure 1 we could know that grandmother is homozygous for the marker and his husband is heterozygous. Their son is affected and homozygous. As a result, we cannot know from whom the disease is inherited for their son that we call as phase-unknown.

The same pedigree as above in figure 2 but a microsatellite is typed. In this situation, the alleles of grandmother and grandfather are different from each other so we can decide the affected son has inherited the disease gene allele marked as C.

2.1.2 SNPs

Single nucleotide polymorphisms (SNPs) occur about 1 in 1,000 bases in the human genome. (Ann-Christine Syvänen 2001, 12) We know that if SNPs located in coding region, changing of SNPs in genes are cause of mostly known mendelian diseases while SNPs in non-coding area have no effect on phenotype. In addition, SNPs are used as makers to identify the disease genes and heterozygous and homozygous SNP genotypes can be gained by PCR detecting restriction fragment length polymorphisms

2.2 Analysis of linkage between markers

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Linkage analysis is a tremendously important and powerful approach in medical genetics. Because it is the only method that allows mapping of, disease genes which are detectable only as phenotypic diseases. (Nussabaum, McInnes, and Willard 2001, 118) Linkage analysis is studying pedigrees in order to know whether two genes are linked when passed down to the next generation. It analyzes markers to identify a region linked to the disease. Genetic variations, in the form of multiple alleles of many genes, exist in most natural populations of organisms.

2.2.1 Recombination Fraction

Recombination Fraction indicates the probability of crossover between two alleles at linked loci if only the linkage phase at two linked loci is known in parents. The crossover is determined by the genetic distance between two allele, which is measured by centiMorgan. This explains why there is 300 evenly distributed polymorphic markers are able to be heterozygous. As human genome include nearly 3000 genes equal to 3000Mb physical distance or 3000cM genetic distance. So 300 markers enable to narrow down the region to 10cM.

2.2.2 Testing for Linkage - LOD score

LOD score of 3.0 demonstrate there is definite evidence of linkage of two loci and it indicates that the likelihood of linkage is 1000 times greater than the likelihood against linkage. LOD score of 2.0 is strong evidence of linkage. On the other hand, LOD score of -2 is the evidence of non-linkage. In practice, we calculate the lod score for each family, and then in order to gain a maximum likelihood estimate of whole family trees, each lod scores are summed to reach the largest total score. Because maximum likelihood estimate is the most likely distance between two loci.

2.3 Narrowing down candidate region

In this family, two children inherit disease from their father and 1 4 3 2 1 chromosome. From child 1 we infer from child 1 that genetic disorder lies between marker 1 and centromere. Moreover, child 2 tells that genetic disorder is in marker 3 to centromere area. It indicate that disease gene locate in this narrow region. So marker 3 is telemeric flanking marker. The more families are studied, the higher probability of narrowing down to a small size.

3. Identifying candidate gene

Contigs can be downloaded from the human genome database. A genome browser Ensembl is used to search for expressed transcripts in the candidate region. The website search displays all known sequences and genes in a specific region of genome. The Ensembl website gives the possibility of directly downloading data, whether it is the DNA sequence of a genomic contig of identifying novel genes in, or positions of SNPs in a gene working on.

4. Confirming a candidate gene- Mutation screening

If candidate gene cause the disease, it must be tested individually to see that if mutations in them do cause the disease or not. Mutations screening is the most classical and popular method so far being used. The reason is that it is generally applicable and comparatively rapid. ( Strachan & Read 2003, 428) The principle of a mutation screening is that the nucleotide sequence of the gene in affected individuals will differ from the sequence content of the same gene in individuals with a normal phenotype.

4.1 Single-strand conformation polymorphism Analysis

Single-strand conformation polymorphism analysis is one of the most widely used procedures for detecting gene mutations as it is simple and cheap. Some or, when feasible, all of the exons of a cloned gene are amplified individually by PCR from the DNA of affected and unaffected individuals.

To do this each pair of primers is tested with whole human DNA to ensure that they amplify only single copy regions. In other words, each pair of primers produces a single band, For optimal results with the SSCP, each primer pair should amplify about 200bp of DNA. Each pair of primers is determined from sequences that flank each exon or from the terminal ends of each exon. In addition, DNA sequence data from genomic clones can be used to develop PCR-based assays to search for mutations in the 5' upstream region adjacent to the first exon, in the 3' downstream region adjacent to the last exon of a gene, and across the splice junctions of exons and introns.

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After PCR amplification of DNA samples from affected and unaffected individuals, the products of each reaction are denatured, rapidly cooled, and electrophoresed. Each denatured single-strand DNA molecule assumes a three-dimensional conformation that is dependent on its primary nucleotide sequence. The conformation is the consequence of intrastrand base pairing and the formation of other bonds. Because of base complementarity, the two single strands of a double-stranded DNA molecule have different nucleotide sequences, and each has a different three-dimensional conformation. The different conformation have different rates of migration in a gel during electrophoresis. Hence, two bands can be visualized by DNA-specific staining, autoradiography, if the strands were radiolabeled during PCR amplification, or fluorescence with fluorescent dye markers.

If two DNA molecules from different sources representing the same segment of a gene differ by a single nucleotide pair, then the conformations of the two single DNA strands from one source are very likely to be different from those of the two strands from the other source. In other words, each of the four strands will migrate during electrophoresis at its own distinctive rate. With side-by-side comparisons between DNA from affected and unaffected individuals, the differences in the migration of single-strand DNA molecules are easily detected. The SSCP localizes a nucleotide alteration to a specific region or exon of a gene. The nature of the mutational difference is not revealed by the SSCP. This information can be obtained by DNA sequencing. The SSCP can detect about 90% of the single base pair mutations in PCR products that are 200bp or less. (Pasternak 2005,191-192)

5. Examples of finding genes of marfan diseases

Marfan syndrome is an autosomal dominant disorder caused by mutations in the gene which encodes fibrillin and affects 1 in 10, 000 people. This is a major component of extracellular microfibrils. ( Young 2005, 107) In 1991, TSIPOURAS et al. (1991, 4486) found that Marfan syndrome is closely linked to a marker on chromosome 15ql.5->q2.1. Twelve three-generation pedigrees were pooled typing Restriction Fragment Length Polymorphism to DNA from all subjects. Five polymorphic marker loci on chromosome

15 were used for analysis of linkage with MFSJ. They discovered that order of four markers are centromere-DJSS48-D15S49-DJ5S45-D15S29-telomere and from linkage study they came to the conclusion that the most possible location of MFSJ is either proximal to D15S48 or between D15S48 and D15S49. Collod et al. (1994) investigated more than 170 subjects and used microsatellite of (AC)n markers to tested for linkage to MFS2 with the MLINK program. Then to confirm the localization of MFS2, 10 polymorphic markers proximal and distal to D3S1300, and spanning a region of 54 cM, were studied: tel-D3S1263, D3S1286, D3S1266, D3S1277, D3S1289, D3S1261, D3S1284, D3S1274, D3S1276, D3S1281-cen. calculating lod scores give definite evidence for marker D3S2336. So they conclude that second locus for Marfan syndrome maps to chromosome 3p24.2-p25.


Venken T. , and J. Del-Favero. 2007. Chasing Genes for Mood Disorders and Schizophrenia in Genetically Isolated Populations. Human Mutation 8(12),1156-1170.

Hartl,Genetics: analysis of genes and genomes/Daniel L.Hartl and Elizabeth W. Jones. 7th Edition Jones and Bartlett Publishers, Inc. Canada printed in USA

Strachan, T., and A. Read. 2003 Human Molecular Genetics3.London: Garland Science

Turnpenny, P., and S. Ellard. 2007. Emery's Elements of Medical Genetics 13th Edition Elsevier Limited Printed in China

Syvänen, A. 2001. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nature Reviews Genetics 2:930-942

Nussabaum, R. J., McInnes, R, and Willard H. 2001. Thompson& Thompson Genetics In Medine 6th Edition. W.B. Saunders Company

Tsipouras P, Sarfarazi M, Devi A, Weiffenbach B, Boxer M. 1991. Marfan syndrome is closely linked to a marker on chromosome 15q1.5----q2.1. Proc Natl Acad Sci U S A. 15; 88(10):4486-8.