HLA-DRB1* FREQUENCIES IN LOMBARDY AND TRENTINO VALLEYS DEFINED BY HIGH RESOLUTION TYPING METHODS

HLA-DRB1* FREQUENCIES IN LOMBARDY AND TRENTINO VALLEYS DEFINED BY HIGH RESOLUTION TYPING METHODS ATTILIO FABIO CRISTALLO 1, NADIA CESCHINI 1, PAOLO GOTTARDI 1, GIULIANA LANDO 2, UMBERTO ROSSI 2, GIAMBATTISTA BERTANI 2, SALVATORE CASSANO 3, GREGORIO SANTORI 4, FEDERICO BAROCCI 4, SERGIO BAROCCI 4 1 Transfusion Medicine and Immunohematology Unit, HLA Laboratory, Santa Chiara Hospital, APSS Trento 2 Transfusion Medicine and Immunohematology Unit, HLA Laboratory, Niguarda Ca Granda Hospital, Milano 3 Biochemistry and Molecular Biology Laboratory, Niguarda Ca Granda Hospital, Milano 4 Department of Surgery and Immunology Unit, San Martino Hospital and University of Genova Abstract Population data on the HLA system are important to several areas of medicine and science. HLA-DRB1* allele frequencies found in 105 and 242 unrelated Italian healthy individuals from Lombardy and Trentino Valleys (from Alto Garda, Val di Ledro, Valli Giudicarie, Val Rendena, Val di Sole, Val di Non, Trento, Val di Fiemme, Val di Fassa, Valsugana, Vallagarina) were estimated, respectively. Sequencing Protrans S4 commercial kit and sequence-specific primers designed to match single alleles were used for specific primer polymerase chain reactions (PCR-SSP) to determine HLA-DRB1* alleles. A total of 30 DRB1* and 37 DRB1* alleles were identified at the 4-digit level in the Lombardy (LRP) and Trentino Valleys population (TrVP) respectively. A comparison between the allelic frequencies of DRB1* locus of the two populations shows that most of these frequencies are shared. One exception is the frequency of DRB1*11:04 allele in the TrVP characterized by a statistically significant lower frequency (3.09% vs. 14.3%, p <0.0138 ) than the LRP. Our data demonstrate that two populations are not genetically different for the DRB1* locus. The present findings as well as being useful for elucidating the genetic background of the populations, can also provide data for HLA matching in clinical transplantation, information about the distribution of HLA genotypes in different populations for the development of peptide-based vaccines and also for paternity diagnosis and evolutionary factors such as genetic drift, migration and selection. Keywords: HLA 4-digit level, DRB1* allele frequencies, Italian populations of Lombardy and Trentino Valleys Introduction The human major histocompatibility complex (MHC) also called human leucocyte antigen (HLA) is located at chromosome 6p21.31. HLA is the most gene-dense region and it plays an important role in the generation of immune response. According to the IMGT (the international ImMunoGene Tics information project - July 2011)/HLA database, a total of 975 HLA-DRB1* alleles, have been identified. The extreme polymorphism of the human leucocyte antigen (HLA) system has been validated as useful tool for distinguishing the origin and the genetic diversity within different ethnic groups but also widely recognized as highly informative in many research studies in different clinical fields since the 1st International HLA Workshop and in all the subsequent International Workshops. Generally, HLA gene frequencies correlate with geographically related populations. In any case, the existence or absence of gene flow among neighbouring ethnic groups may be assessed with the study of HLA allele frequencies and the corresponding distances and haplotypes. As for Italy, there are many reports using Italian populations on HLA class I and class II antigen distribution, due to their frequent involvement in several autoimmune diseases. However, the majority of these studies refer to HLA- B* and DRB1* alleles detected by PCR-SSP low resolution, whereas only a few, which are restricted to certain Italian provinces and/or Italian regions, have so far been available for the analysis of the molecular diversity of HLA class I and class II alleles by high resolution typing DNA methods. Recently, implementation of these techniques has allowed a significantly wider spectrum of variation to be detected including rare alleles and particular haplotypes and has made HLA comparisons a more discriminative tool to further clarify population relationships. As frequency distributions of HLA alleles differ significantly among populations belonging to different areas of Italy which represent an admixture of the primitive individuals, knowledge of the allele frequency distributions in Italian regions but also history with its various waves of migration along the peninsula since prehistory, can shed light on the evolution of HLA polymorphisms as well as on the evolution and 50

migration of Italian populations. Italy, among the studied nations, presents an evident genetic differentiation of northern from southern regions with Sardinia being quite far apart. In order to add additional data in these surveys, we examined here the populations of Lombardy and Trentino (from Alto Garda, Val di Ledro, Valli Giudicarie, Val Rendena, Val di Sole, Val di Non, Trento, Val di Fiemme, Val di Fassa, Valsugana, Vallagarina), respectively. Lombardy is bordered by Switzerland (north: Canton Ticino and Canton Graubunden) and by Italian regions Emilia-Romagna (south) Trentino- Alto Adige/Sudtirol and Veneto (east) and Piemont (west). The capital is Milan (historic, cultural and artistic city). Since the end of World War II, Milan has been host to two waves of mass migration: the first, workers from within Italy; the second, immigrants from outside the peninsula. By the end of the 1990s Milan had a 10 per cent foreign immigrant population, the vast majority of whom worked in the low-level service sector (restaurant workers, cleaners, maids, domestic workers) or in factories. In January 2009, the Italian national institute of statistics ISTAT estimated that 181393 foreign-born immigrants lived in Milan, representing the 14% of the total population. About 10 million people live in Lombardy (16.2% of the national population, 2% of the European Union population). Lombardy was inhabited by Celtic peoples from the 5th century BC and it was conquered by Rome after the Second Punic War (218-201 BC), upon which it became part of Cisalpine Gaul. The region suffered heavily in the barbarian invasions that ended the Western Roman Empire, and from A.D. 568 to 774 it was the centre of the kingdom of the Lombards, a Germanic people who gave their name to the region. The Lombard kingdom ended in 774, and Lombardy became part of the empire of the Frankish king Charlemagne. Frankish rule continued until 887, and after the breakup of the Carolingian empire a number of independent units, mostly towns ruled by counts or bishops, emerged in Lombardy. These towns growing prosperity by the 11 th century was based on the role of the middle Po river valley as a transit point for trade between the Mediterranean and the trans-alpine lands. Trentino is one of the two provinces which make up the region of Trentino-Alto Adige/Südtirol, which itself is an autonomous region. The region is bordered by Tyrol (Austria) to the north, by Graubünden (Switzerland) to the north-west and by the Italian regions of Lombardy and Veneto to the west and south, respectively. Trentino is an almost entirely mountainous region with a main valley crossing it in its centre. This valley is named after the Adige river flowing within it. The region is divided into two autonomous provinces: Trentino (Province of Trento) and South Tyrol (Province of Bolzano). The province of Trento has a total population of 524.826 (2010). The region was conquered by the Romans in 15 BC. After the end of the Western Roman Empire, it was divided between the invading German tribes in the Lombard Duchy of Tridentum (today s Trentino), the Alamannic Vinschgau and the Bavarians taking the remaining part. After the creation of the Kingdom of Italy under Charlemagne, the Marquisate of Verona included the southern areas of Bolzano, while the Duchy of Bavaria received the remaining part. The principal towns of Trentino lay on the Adige Valley as it is the largest one and has been a historical passage connecting Italy with Northern Europe. The aim of this study was to analyze the allele frequencies of HLA-DRB1* (4 digits typed) of 105 and 244 unrelated healthy individuals at least three generations from Lombardy and Trentino (from Alto Garda, Val di Ledro, Valli Giudicarie, Val Rendena, Val di Sole, Val di Non, Trento, Val di Fiemme, Val di Fassa, Valsugana, Vallagarina), respectively. This analysis may contribute to the development of an updated and more detailed study on the polymorphism of HLA genes and may represent a useful tool for comparison with the HLA gene and haplotype frequencies of other Italian and European populations at high resolution. Materials and methods Population samples Blood samples collected from 105 and 244 unrelated Italian healthy individuals of both sexes that had all at least four grand-parents born in Lombardy Region (Northern Italy) and Trentino Valleys (from Alto Garda, Val di Ledro, Valli Giudicarie, Val Rendena, Val di Sole, Val di Non, Trento, Val di Fiemme, Val di Fassa, Valsugana, Vallagarina), respectively, were used to estimate the frequencies of HLA-DRB1* locus. HLA genotyping, DNA sequencing and statistics All HLA genotypings are the result of methods required to resolve to the 4-digit allele level within the antigen binding domain (Cano et al, 2007). Genomic DNA was isolated from whole blood or buffy-coats by using a GenoNTM-6 automatic nucleic acid purification and an ipreptm purification instrument. Commercial kits with sequence-specific primers designed to match single alleles were used for specific primer polymerase chain reactions (PCR- SSP) (Olerup SSP-genotyping and Protrans SSP kits), to determine HLA-DRB1* alleles (Aldener-Cannavá et al, 2001; Olerup and Zetterquist, 1991; Olerup and Zetterquist, 1992; Welsh and Bunce, 1999). Single allele specific amplification and sequencing were also used separately, by using a Protrans S4 commercial kit (Protrans, Germany). All DNA sequences were detected on automated fluorescent 51

DNA sequencers (Applied Biosystems ABI Prism 3130 and Applied Biosystems ABI 3500Dx Genetic Analyzers) (Blasczyk, 2003; Sanger et al, 1977; Voorter et al, 1997). Data were processed with Helmberg Score SSP, Assign 3.5 plus Conexio Genomics and Sequence Pilot 3.4 software. HLA database were continuously updated in line with the IMGT/HLA databases. Human leucocyte antigen typing results were obtained at high resolution so that all statistical were performed at the four-digit allele family level. The phenotype frequencies (PF) were calculated by direct counting method. The loci where only one allele was detected were considered to be homozygous for that allele (Bodmer and Bodmer, 1970; Müller et al, 2003; Schipper et al, 1997; Schipper et al, 1998). Gene frequencies (GF) of HLA alleles and their Standard Errors (SE) were calculated as previously reported (Pickbourne et al, 1978). The chi square test or when required, Fisher s exact test, was used for comparison of the allele frequencies between LRP and TrVP populations. The threshold of significance for p values was 0.05. The allelic frequencies at DRB1* locus were in Hardy- Weinberg Equilibrium for both populations. Results and discussion Genotyping of HLA-DRB1* alleles were performed from 105 and 244 unrelated Italian healthy individuals of both sexes that had all at least four grand-parents born in Lombardy Region and Trentino Valleys. Our data refer exclusively to the populations born in Northern Italy. Newborns whose parents or grandparents were non Caucasian or who were not born in Italy were excluded from the study. Molecular analysis of the Lombardy Region population (LRP) samples determined 30 allele groups for gene HLA-DRB1*, the most common (frequency >5%) being DRB1*11:01 (27.6%), DRB1*07:01 (23.8%), DRB1*03:01 (19.0%), DRB1*01:01 (17.0%), DRB1*11:04 (14.3%), DRB1*15:01 (10.4%), DRB1*13:02 (8.6%), DRB1*11:03 (7.6%), DRB1*14:01 (6.7%), DRB1*14:54 (6.7%), DRB1*13:01 (5.7%), DRB1*10:01 (5.7%), DRB1*12:01 (5.7%) and DRB1*16:01 (5.7%). Table 1 lists all the allele groups observed for this gene in the LRP. In the case of the Trentino Valleys population (TrVP), 37 allele groups were detected, including the following most frequent allele groups (frequency >5%) HLA-DRB1* locus Number observed Phenotype frequency (%) Gene frequency Standard Error DRB1*01:01 18 17.0 0.089 0.021 DRB1*01:02 4 3.8 0.02 0.01 DRB1*03:01 20 19.0 0.10 0.022 DRB1*04:01 4 3.8 0.02 0.01 DRB1*04:02 1 0.95 0.005 0.005 DRB1*04:03 2 1.9 0.01 0.007 DRB1*04:05 3 2.8 0.015 0.008 DRB1*04:07 4 3.8 0.02 0.01 DRB1*07:01 25 23.8 0.127 0.025 DRB1*08:02 1 0.95 0.005 0.005 DRB1*08:04 1 0.95 0.005 0.005 DRB1*09:01 1 0.95 0.005 0.005 DRB1*10:01 6 5.7 0.03 0.012 DRB1*11:01 29 27.6 0.15 0.026 DRB1*11:02 2 1.9 0.01 0.007 DRB1*11:03 8 7.6 0.039 0.014 DRB1*11:04 15 14.3 0.075 0.019 DRB1*11:34 1 0.95 0.005 0.005 DRB1*12:01 4 3.8 0.02 0.01 DRB1*13:01 6 5.7 0.03 0.012 DRB1*13:02 9 8.6 0.044 0.014 DRB1*13:03 4 3.8 0.02 0.01 DRB1*14:01 7 6.7 0.034 0.013 DRB1*14:02 2 1.9 0.01 0.007 DRB1*14:04 1 0.95 0.005 0.005 DRB1*14:54 7 6.7 0.034 0.013 DRB1*15:01 11 10.4 0.054 0.016 DRB1*15:02 5 4.7 0.024 0.011 DRB1*16:01 6 5.7 0.03 0.012 DRB1*16:02 1 0.95 0.005 0.005 Tab.1 HLA-DRB1* gene frequencies seen in the Lombardy Region population (n =105) 52

DRB1*03:01 (11.98%), DRB1*11:01 (11.36%), DRB1*07:01 (11.15%), DRB1*13:01 (8.47%), DRB1*15:01 (8.47%) and DRB1*01:01 (6.2%). Table 2 shows the complete list of HLA-DRB1* allele groups observed in the sample from the TrVP. Table 3 shows the comparison of allele frequencies of DRB1 * locus between TrVP and LRP. Comparing the allelic frequencies reported in both populations no statistically significant difference was highlighted for almost all the alleles, excluding DRB1*11:04, characterized by lower allelic frequency in the TrVP ( 3.09% vs 14.3 %, p< 0.01389) than that of LRP. DRB1*11:04 is also reported to be predominant over 11:01 in France and Germany (Matullo et al, 1994) but also in Greece and Romania whereas studies in Crete, Slovenia, Croatia show equal or reversed frequencies (Arnaiz-Villena et al, 1999; Grubic et al, 1995; Reed et al, 1992). Like in other European populations, other alleles such as DRB1*01:01, DRB1*03:01, DRB1*07:01 and DRB1*11:01 (Clayton and Lonjou, 1997) also occur frequently in LRP and TrVP, respectively (Tables 1 and 2). Analysis of allelic frequencies seems to confirm that the TrVP is not genetically different from LRP but also that these two populations appear to be genetically similar to other European populations at least on the DRB1*locus. The extreme degree of allelic polymorphism of the HLA system makes it an important topic for population studies. It is well established that the frequency of many HLA alleles differs remarkably between various ethnic groups. The knowledge and comparison of its degree of polymorphism among each population from different Italian regions and in different populations in the world can provide valuable information in many fields such as HLA-DRB1* locus Number observed Phenotype frequency (%) Gene frequency Standard Error DRB1*01:01 30 6.2 0.0315 0.008 DRB1*01:02 12 2.47 0.0125 0.004 DRB1*01:03 3 0.63 0.0032 0.002 DRB1*03:01 58 11.98 0.062 0.011 DRB1*04:01 21 4.33 0.022 0.067 DRB1*04:02 1 0.21 0.0011 0.001 DRB1*04:03 13 2.68 0.0135 0.005 DRB1*04:04 15 3.09 0.0156 0.006 DRB1*04:05 3 0.63 0.0032 0.0024 DRB1*04:06 1 0.21 0.0011 0.001 DRB1*04:07 1 0.21 0.0011 0.001 DRB1*04:08 2 0.42 0.0022 0.0021 DRB1*07:01 54 11.15 0.0574 0.0104 DRB1*08:01 22 4.54 0.023 0.0068 DRB1*08:02 1 0.21 0.0011 0.001 DRB1*08:04 2 0.42 0.0022 0.0021 DRB1*09:01 1 0.21 0.0011 0.001 DRB1*10:01 8 1.65 0.0083 0.004 DRB1*11:01 55 11.36 0.0585 0.0109 DRB1*11:03 7 1.44 0.0073 0.0038 DRB1*11:04 15 3.09 0.0156 0.006 DRB1*11:10 1 0.21 0.0011 0.001 DRB1*11:15 1 0.21 0.0011 0.001 DRB1*11:27 1 0.21 0.0011 0.001 DRB1*12:01 7 1.44 0.0073 0.0038 DRB1*13:01 41 8.47 0.043 0.0093 DRB1*13:02 16 3.3 0.0167 0.006 DRB1*13:03 3 0.63 0.0032 0.002 DRB1*13:36 1 0.21 0.0011 0.001 DRB1*14:01 6 1.23 0.0062 0.0034 DRB1*14:02 2 0.42 0.0022 0.0021 DRB1*14:04 1 0.21 0.0011 0.001 DRB1*14:54 23 4.75 0.024 0.007 DRB1*15:01 41 8.47 0.043 0.0093 DRB1*15:02 3 0.63 0.0032 0.002 DRB1*16:01 11 2.27 0.0115 0.0048 DRB1*16:02 1 0.21 0.0011 0.001 Tab. 2 HLA-DRB1* gene frequencies seen in the Trentino Valleys population (n =242) 53

anthropology, archaeology, evolution genetics and history. For this type of study, international collaborations are necessary as far as the International Histocompatibility Workshops that provide an excellent opportunity to accumulate a large amount of comparative data on different ethnic groups investigated. Collaborative efforts like this one engage a certain importance because interchanges among populations are becoming more frequent year after year and the genetic traits of many populations especially ethnic minorities will get lost very soon. The present study can be useful for paternity diagnosis in forensic sciences and provide basic and valuable data for anthropological analysis, HLA-associated disease susceptibility and population genetics such as genetic drift, migration and selection. In addition, these population data can also be used in stem cell donor search in order to facilitate selection of acceptable donors for those patients who require stem cell and solid organ transplants, respectively. In conclusion, this is the first report addressing HLA specificity frequencies for the DRB1* locus defined by high resolution typing methods (4-digit level) focused on the population of Lombardy and Trentino Valleys. Both populations show to share almost the same genetic background in agreement with the geographical situation and the historical composition. Phenotype Gene Phenotype Gene HLA-DRB1* locus frequency (%) frequency frequency (%) frequency P value in LRP (n =105) in LRP (n =105) in TrVP (n =242) in TrVP (n =242) DRB1*01:01 17.0 0.089 6.2 0.031 NS DRB1*01:02 3.8 0.02 2.47 0.012 NS DRB1*01:03 0.0-0.63 0.003 DRB1*03:01 19.0 0.10 11.9 0.062 NS DRB1*04:01 3.8 0.02 4.33 0.022 NS DRB1*04:02 0.95 0.005 0.21 0.001 NS DRB1*04:03 1.9 0.01 2.68 0.013 NS DRB1*04:04 0.0-3.09 0.016 DRB1*04:05 2.8 0.015 0.63 0.003 NS DRB1*04:06 0.0-0.21 0.001 DRB1*04:07 3.8 0.02 0.21 0.001 NS DRB1*04:08 0.0-0.42 0.002 DRB1*07:01 23.8 0.127 11.2 0.057 NS DRB1*08:01 0.0-4.54 0.023 DRB1*08:02 0.95 0.005 0.21 0.001 NS DRB1*08:04 0.95 0.005 0.42 0.002 NS DRB1*09:01 0.95 0.005 0.21 0.001 NS DRB1*10:01 5.7 0.03 1.65 0.008 NS DRB1*11:01 27.6 0.15 11.36 0.058 NS DRB1*11:02 1.9 0.01 0.0 - DRB1*11:03 7.6 0.039 1.44 0.007 NS DRB1*11:04 14.3 0.075 3.09 0.016 0.0138 DRB1*11:10 0.0-0.21 0.001 DRB1*11:15 0.0-0.21 0.001 DRB1*11:27 0.0-0.21 0.001 DRB1*11:34 0.95 0.005 0.0 - DRB1*12:01 3.8 0.02 1.44 0.007 NS DRB1*13:01 5.7 0.03 8.47 0.043 NS DRB1*13:02 8.6 0.044 3.3 0.017 NS DRB1*13:03 3.8 0.02 0.63 0.003 NS DRB1*13:36 0.0-0.21 0.001 DRB1*14:01 6.7 0.034 1.23 0.006 NS DRB1*14:02 1.9 0.01 0.42 0.002 NS DRB1*14:04 0.95 0.005 0.21 0.001 NS DRB1*14:54 6.7 0.034 4.75 0.024 NS DRB1*15:01 10.4 0.054 8.47 0.043 NS DRB1*15:02 4.7 0.024 0.63 0.003 NS DRB1*16:01 5.7 0.03 2.27 0.012 NS DRB1*16:02 0.95 0.005 0.21 0.001 NS Tab. 3 Comparison of frequencies of HLA-DRB1* alleles in the Lombardy Region (LRP) and Trentino Valleys (TrVP) populations. NS= not significant 54