MODULE NO.14: Y-Chromosome Testing

Transcription

1 SUBJECT Paper No. and Title Module No. and Title Module Tag FORENSIC SIENCE PAPER No.13: DNA Forensics MODULE No.21: Y-Chromosome Testing FSC_P13_M21

2 TABLE OF CONTENTS 1. Learning Outcome 2. Introduction: Y- Chromosome 3. Applications of Chromosome Y Testing 3.1 Advantages of Y-Chromosome testing 3.2 Limitations of Y- Chromosome testing 4. Structure of Y-Chromosome 5. Human Y-Chromosome Genome 6. Profiling systems 7. Gender Typing 8. Summary

3 1. Learning Outcomes After studying this module, you shall be able to know about- Application of Y- Chromosome testing Structure of Y- Chromosome Different profiling systems for Y-Chromosome testing Gender Typing 2. Introduction: Y-Chromosome 3. The Y chromosome is inherited from the father and is conceded on to all male offspring. Thus, the Y chromosome is distinctive to males. The chromosome encodes dozens of genes essential for male- specific functions, including sex determination and spermatogenesis. Y chromosome loci are very important for forensic DNA profiling and in this module we shall be discussing about the various such applications related to Y chromosome testing. For instance, the Y chromosome STR (Y-STR) used in forensic DNA testing is malespecific and is thus useful in investigations related to sexual assault where male suspects are involved. The evidence that is collected in such cases usually consists of mixtures with high levels of female DNA and low levels of male DNA. The Y chromosome specific loci can thus be examined without interference from the large amounts of female DNA. Furthermore, the Y-STR system is useful for determining the numbers of male perpetrators involved in sexual assault cases involving more than one male. The Y-STR loci used for forensic applications are located in the non-recombining section of the Y chromosome so that paternal lineages can be established. The technique can be used for paternity testing and identification of missing persons. Finally, data interpretation can be simplified by the use of a single allele per Y-STR locus profile.

4 Figure 1: Pedigree showing patrilineal inheritance where all shaded males have the same Y chromosome barring any mutations. 3. Applications of Chromosome Y testing Y chromosome DNA testing is vital for a number of diverse applications of human genetics including forensic evidence examination, paternity testing, historical investigations, studying human migration patterns throughout history, and genealogical research. In terms of forensic applications, there are both advantages and limitations to Y-chromosome testing Advantages of Y- Chromosome Testing: Male-specific amplification can enable examination of a male perpetrator s profile even in mixtures with high levels of female DNA in sexual assault cases Additional mixtures may possibly be analyzed (e.g. fingernail scrapings, saliva on skin, etc.)

5 6. 3. Paternal transmission from a father to all of his sons extends possible reference sample providers and enables tracing family lineages Limitations of Y- Chromosome Testing: Since paternal relatives are indistinguishable, Y-STR typing cannot be used to differentiate among siblings or even distant paternal relatives. 2. Without recombination between loci, the product rule cannot be used and thus the discrimination power of Y-STRs is limited by the size of the population database used. 3. Duplications and deletions can cause difficulties in the analysis. 9. The primary value of the Y-chromosome in forensic DNA testing is that it is found only in males. The SRY (sex-determining region of the Y) gene determines maleness. With ChrY tests, interpretable results can be obtained in some cases where autosomal tests are limited by the evidence, such as high levels of female DNA in the presence of minor amounts of male DNA. These situations include sexual assault evidence from azospermic or vasectomized males and blood blood or saliva blood mixtures where the absence of sperm prevents a successful differential extraction for isolation of male DNA. In addition, the number of individuals involved in a gang rape may be easier to decipher with Y- chromosome results than with highly complicated autosomal STR mixtures. Using ChrYspecific PCR primers can improve the chances of detecting low levels of the perpetrator s DNA in a high background of a female victim s DNA. Y-chromosome testing have been employed on the male individuals who are amelogenin-y deficient so as to verify the mentioned problem. The similar feature of the Y-chromosome that gives it a benefit in forensic testing, namely maleness, is also its principal limitation. A bulk of the Y-chromosome is transferred directly from father to son without recombination to shuffle its genes and provide greater genetic variety to future generations. Random mutations are the only mechanisms for variation over time between paternally related males.

6 Thus, while exclusions in Y-chromosome DNA testing results can aid forensic investigations, a match between a suspect and evidence only means that the individual in question could have contributed the forensic stain as could a brother, father, son, uncle, paternal cousin, or even a distant cousin from his paternal lineage. Needless to say, inclusions with Y-chromosome testing are not as meaningful as autosomal STR matches from a random match probability point-of-view. 10. On the other hand, the presence of relatives having the same ChrY expands the number of possible reference samples in missing person s investigations and mass disaster victim identification efforts. ChrY testing also aids familial searching. Deficient paternity tests where the father is dead or unavailable for testing are benefited if ChrY markers are used. However, an Autosomal DNA test is always preferred when possible since it provides a higher power of distinction. Through the male lineages the Y-chromosome testing has been utilized so as to trace the historical patterns of human migration. Anthropological, historical, and genealogical questions can be answered through ChrY results. Use Forensic casework on sexual assault evidence Verification of amelogenin Y deficient males Paternity Testing Missing persons investigations Human migration and evolutionary studies Historical and genealogical research Advantage Male-Specific amplification (can avoid differential extraction to separate sperm and epithelial cells) Analysis of multiple regions along Y chromosome that should not be affected by deletion of the amelogenin region Male children can be tied to fathers in motherless paternity cases or testing of male relatives if father is unavailable Patrilineal male relatives may be used for reference samples Lack of recombination enables comparison of male individuals separated by large periods of time Surnames usually retained by males; can make links where paper trail is limited

7 4. Y-chromosome Structure A detailed analysis of the finished reference Y chromosome sequence was described in the 19 June 2003 issue of Nature by researchers from the Whitehead Institute and Washington University. At 50Mb, the Y chromosome is the third smallest human chromosome only slightly larger than chromosome 21 (47Mb) and chromosome 22 (49Mb). Figure 2: Structure of Human Y-Chromosome The non-recombining region of the human Y chromosome (NRY) comprises approximately 95% of the chromosome. The two tips of the Y chromosome, known as pseudo-autosomal regions (PAR), recombine with X chromosome homologous regions. PAR1 located at the tip of the short arm (Yp) of the Y chromosome is approximately 2.5Mb in length while PAR2 at the tip of the long arm (Yq) is less than 1Mb in size. Skaletsky et al. (2003) renamed the NRY the male-specific region (MSY) because of evidence of frequent gene conversion or intra-chromosomal recombination. A total of 156 known transcription units including 78 protein-coding genes are present on MSY.

8 The Y chromosome is highly duplicated either with itself or with the X chromosome. Three classes of sequences have been characterized in the Y chromosome: X-transposed, X-degenerate, and ampliconic. 5. Human Y-chromosome Genome The human Y chromosome genome contains approximately 60 million bp and the chromosome can be divided into two regions: the pseudo- autosomal region (PAR) and the male- specific Y (MSY) region. 1. Pseudo-Autosomal Region: Approximately 5% of the Y chromosome sequence is located at the telomeres of the chromosome. In particular, PAR1 is located at the tip of the short arm and PAR2 is located at the tip of the long arm. This region undergoes recombination with homologous region on the X chromosome during meiosis in males. Figure 3: a. Schematic of X and Y sex chromosomes. b. The Y chromosome is composed of both Euchromatic and Heterochromatic region.

9 2. Male-Specific Y Region: The remainder of the Y chromosome is known as MSY region. It was previously called the non-recombining Y (NRY) region. It does not participate in homologous recombination. However, certain sections involve intra-chromosomal gene conversion. About 40 megabases (Mb) within the MSY region are heterochromatic (highly repetitive sequences) including the centromeric region and the bulk of the distal long arm. The euchromatic region is about 23 Mb and most of it has been sequenced. Certain sections of the euchromatic region share some homology with the X chromosome. For instance, X transposed sequences of the Y chromosome are 99% identical to sequences within Xq21 (a band in the long arm of the X chromosome). Additionally, dozens of genes located in the euchromatic region share 60% to 96% homology with their X chromosome counterparts. These X- homologous regions should be avoided when selecting chromosome- specific markers for DNA profiling. 3. Polymorphic Sequences: The Y chromosome contains an abundance of repetitive elements, namely STRs, Alu, and LINE elements. Many of these are highly polymorphic. To date Y-STRs are usually used for Y chromosome DNA testing. Single nucleotide polymorphisms (SNPs) at the Y chromosome are also useful for forensic applications. 6. Profiling Systems 1. Y-STR More than 400 STR loci have been identified in the Y chromosome genome. The precise locations of these loci have been sequentially mapped using human genome sequencing data. The distribution of Y-STR loci at the Y chromosome has also been analyzed. Most Y-STR loci, approximately 60% of the 400 identified, are located at the long arm of the chromosome; about 22% are located at the short arm and a few are found in the centromeric region.

10 Y-STRs in the telomeric region have yet to be identified. Y-STRs have been analyzed. Among 400 Y-STRs, 60% are dimeric repeats, 39% are trimeric, 45% are tetrameric, 9% are pentameric, and 1% is hexameric. Fewer than half the STRs have been characterized. Some loci are polymorphic and are useful for forensic applications and developing new Y-STR multiplex systems. The STR loci at the Y chromosome are usually referred to as haplotypes. A haplotype is a collection of alleles that are usually linked (inherited together) since homologous recombination does not occur on the majority of the Y chromosome. The most commonly used Y-STR loci for forensic testing are: DYS19, DYS385a and b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393. Figure 4: Human Cytogenetic map of Y-Chromosome. The Y-STRs and positions are shown.

11 2. Core Y-STR Loci: In 1997, the European minimal haplotype (EMH) locus set was recommended by the International Y-STR User Group for forensic applications. This haplotype set includes a core set of Y-STR loci: DYS19, DYS385a and b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393. In 2003, the U.S. haplotype loci were recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) for forensic DNA analysis. The U.S. haplotype loci include the EMH loci set plus two additional loci, DYS438 and DYS439. DYS385 and DYS389 are multi-local Y-STR loci (MLL).The MLL designation reference to a presence of a particular STR at more than one site on the Y chromosome DNA due to duplication. To date, about 50 such MLL Y-STRs have been identified. Further MLL subdivisions are designated are bi-local, tri-local, etc. DYS385 and DYS389 are bi-local. The DYS385 locus has two inverted duplicated clusters and is separated by a 4x10 4 bp interstitial region. It can be amplified by a single set of primers. One allele is observed if the duplicates are the same length. If the duplicated clusters have different lengths, they can generate two different alleles when amplified. The smaller sized allele is designated a and the larger sized allele is designated b. The DYS389 locus has two duplicated clusters with the same orientation. In a single set of PCR primers, there are two binding sites for the same forward primer at each 5 flanking sequence of the core repeat region of DYS389. These binding sites between DYS389I and DYS389II are about 120bp apart. Therefore, two amplicons are produced. DYS389I is designed for the smaller allele and DYS389II is designated for the larger allele.

12 The average mutation rate for the core Y-STR loci is approximately 10-3 per generationsimilar to the mutation rate of autosomal STR loci. Mutations can exert major impacts on the interpretation of paternity test results. Figure 5: (a) DYS385a/b PCR primer binding site (b) DYS 389 I/II PCR primer binding site The figure above shows a schematic illustration of how PCR Primer binding sites give rise to multi-copy PCR products for (a) DYS385a/b and (b) DYS 389 I/II. Arrows represent either forward F or reverse R primers. In case of DYS385a/b, the entire region around the STR repeat is duplicated and spaced about 40,775 bp apart on the long arm of the Y chromosome. Thus, amplification with a single set of primers gives rise to one peak if the a repeat region is equal in the size to the b repeat region or separate peaks if a and b differ in length. DYS389 possesses two primary repeat regions that are flanked on one side by a similar sequence. Widely used forward primers bind adjacent to both repeats generating amplicons that differ in size by approximately 120bp.

13 3. Multiplex Y-STR The application Y-STR for forensic casework was initiated in Europe. In the U.S., the laboratory of the Office of Chief Medical Examiner in New York City was the first to perform Y-STR testing of four loci (DYS19, DYS390, DYS389I and II). The use of Y- STR loci has been facilitated by various commercially available PCR amplification kits in multiplex systems. ReliaGene Technologies developed the first commercial multiplex Y-STR system, the Y- PLEX TM 6. The kit includes DYS19, DYS385a and b, DYS389II, DYS390, DYS391 and DYS393. Additional commercially available kits with more Y-STR loci are now available and have been validated for forensic use. To improve discriminating power, multiplex systems including new Y-STR loci are desired. Many new Y-STR loci are being characterized for developing new multiplex systems. Figure 6: Result from the commercial Y-STR kit Y-PLEX TM 6

14 7. Gender Typing Gender typing of a biological sample is useful in forensic investigation, for example, for victim identification in disaster cases and suspect identification in sexual assaults. One commonly used gender typing marker is the amelogenin (AMEL) locus Amelogenin Locus This region encodes extracellular matrix proteins involved in tooth enamel formation. Mutations to the AMEL gene can lead to an enamel defect known as amelogenesis imperfect. The AMEL locus has two homologous genes: AMELX (Xp22.1-Xp22.3) is located on the human X chromosome and AMELY (Yp11.2) is located on the human Y chromosome. Although the genes constitute a homologous pair, they differ in size and sequence. Gender typing can be performed using various primers designed specifically for the sequences of the homologous region on these genes, followed by amplification. Different sizes of amplicons are obtained. The most commonly used gender typing method at the AMEL locus is the detection of a 6-bp deletion at intron 1 of AMELX. This deletion is not present in AMELY. Primer sets were developed to amplify both alleles in a single PCR by Forensic Science Service in the United Kingdom in The amplicons generated from AMELY and AMELX are separated by electrophoresis. The observation of the AMELX fragment also indicates a female, whereas the observation of both AMELX and AMELY indicates a male. Nevertheless, primate and some rudiment DNA can be amplified as well but the amplicon sizes vary. The AMEL locus has been co-amplified with other markers to provide a combined gender and identity test. Such combined tests have been used in D1S80 AFLP and various STR multiplex analyses.

15 AMELY Null Mutations Several cases of AMELY null mutations have been reported. Only the AMELX fragment was detected in these AMELY null males. Many of them are phenotypically normal but present the AMEL gender types of females. Various interstitial deletions at the Y chromosome short arm have been identified as the cause of some AMELY null gender typing. The frequency of AMELY null males is rare, but is higher in Sri Lanka and India. 8. Summary In this module, we learnt about the Y-Chromosome, its structure. Applications of Y-Chromosome testing like Forensic casework on sexual assault evidence, Verification of amelogenin Y deficient males, Paternity testing, Missing persons investigations, human migration and evolutionary studies. Human y- Chromosome Genome like the pseudo-autosomal Region, Male- Specific Y Region, Polymorphic Sequences. Different Profiling systems used for Y chromosome such as Y-STR, Core Y-STR Loci, Multiplex Y-STR Gender typing is also used in forensic investigation such as Identification in mass disasters, and suspect identification in cases of gang rapes.