Genomic sequencing for newborn screening: current perspectives and challenges.

Traditional newborn screening (NBS) serves as a critical tool in identifying conditions that may impact a child's health from an early stage. Newborn sequencing (NBSeq), the comprehensive analysis of an infant's genome, holds immense promise for revolutionizing health care throughout the lifespan. NBSeq allows for early detection of genetic disease risk and precision personalized medicine. The rapid evolution of DNA sequencing technologies and increasing affordability have spurred numerous endeavors to explore the potential of whole-genome sequencing in newborn screening. However, this transformative potential cannot be realized without challenges. Ethical aspects must be carefully navigated to safeguard individual rights and maintain public trust. Moreover, genomic data interpretation poses complex challenges due to its amount, the presence of variants of uncertain significance, and the dynamic nature of our understanding of genetics. Implementation hurdles, including cost, infrastructure, and specialized expertise, also present barriers to the widespread adoption of NBSeq. Addressing these challenges requires collaboration among clinicians, researchers, policymakers, ethicists, and stakeholders across various sectors. Robust frameworks for informed consent, data protection, and governance are essential. Advances in bioinformatics, machine learning, and genomic interpretation are crucial for translation into actionable clinical insights. Scalability and improving downstream health care access are vital for equitability, particularly in underserved communities. By fostering interdisciplinary collaboration, advancing technology and infrastructure, and upholding ethical principles, we can unlock the full potential of NBSeq as a tool for precision medicine and pave the way toward a future where every child has the opportunity for a healthier, genomics-informed start to life.

Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war - a 30-year review.

Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.

Genetic sub-structuring of Croatian island populations in the Southeastern European context: a meta-analysis.

AIM: To use the method of meta-analysis to assess the influence of island population isolation on the sub-structuring of the Croatian population, as well as the influence of regional population groups on the sub-structuring of the Southeastern European population with regard to basic population genetic statistical parameters calculated by using STR locus analysis. METHODS: Bio-statistical analyses were performed for 2877 unrelated participants of both sexes from Southeastern Europe. Nine autosomal STR loci (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, and D7S82) were analyzed by using standard F-statistics and population structure analysis (Structure software). RESULTS: Genetic differentiation of Croatian subpopulations assessed with the FST method was higher at the level of the Croatian population (0.005) than at the level of Southeastern Europe (0.002). The island of Vis showed the most pronounced separation in the Croatian population, and Albanians from Kosovo in the population of Southeast Europe, followed by Croatia, Bosnia and Herzegovina, and Hungary. CONCLUSION: The higher structure of Croatian subpopulations in relation to Southeastern Europe suggest a certain degree of genetic isolation, most likely due to the influence of endogamy within rural island populations.

Influence of genetic substructuring of statistical forensic parameters on genetic short tandem repeat markers in the populations of Southeastern Europe.

AIM: To investigate the influence of specific intrapopulation genetic structures on interpopulation relationships. Special focus was the influence of island population isolation on the substructuring of the Croatian population, and the influence of regional population groups on the substructuring of Southeast European populations. METHODS: Autosomal short tandem repeat (STR) loci were analyzed by using four forensic parameters: matching probability (PM), power of discrimination (PD), power of exclusion (PE), and polymorphic information content (PIC) on a sample of 2877 unrelated participants of both sexes. A sample set comprising 590 participants was analyzed for the first time, and 2287 participants were included from previous studies. The analysis was performed with PowerStats v. 1.2. RESULTS: The analysis of forensic parameters for all nine loci in the Croatian subpopulations showed the largest deviations in the populations of the islands of Korcula and Hvar. The smallest deviations were found in the mainland population. As for Southeast European populations, the largest deviations were found in the population of North Macedonia, followed by Romania, Albanians from Kosovo, and Montenegro, while the smallest deviations were found in the population of Hungary. CONCLUSION: The comparison of forensic parameters between different subpopulations of Croatia and Southeast Europe indicates that the isolation of individual Croatian subpopulations and rare alleles in their gene pool affect the values of forensic parameters. Specific features of (sub)populations should be taken into account for appropriate sampling of the total population when creating a DNA database of STR markers.

Croatian genetic heritage: an updated Y-chromosome story.

AIM: To analyze an additional set of ˝Y-chromosome genetic markers to acquire a more detailed insight into the diversity of the Croatian population. METHODS: A total of 518 Yfiler Plus profiles were genotyped. Allele frequencies, haplotype frequencies, and haplotype diversity were calculated by using the STRAF software v. 2.0.4. Genetic distances were quantified by Rst with AMOVA online tool from the YHRD. The evolutionary history was inferred with the neighbor-joining method of phylogenetic tree construction in the MEGAX software. Whit Athey's Haplogroup Predictor v. 5 was used for additional comparison with regional and other European populations. RESULTS: A total of 507 haplotypes were used for genetic STR analysis. An interpopulation study on 17 Y-STR markers showed the lowest genetic diversity between the Croatian and Bosnian-Herzegovinian populations and the highest between the Croatian and Irish populations. Additional interpopulation comparison with the original 27 Y-STR markers (for the population with available data) was also performed. A total of 518 haplotypes were used in the determination of haplogroup diversity. Haplogroup I with its sublineage I2a expressed the highest prevalence. The second most prevalent haplogroup was R, with its major sublineage R1a, except for the subpopulation of Hvar, where E1b1b was the second most prevalent haplogroup. Rare haplogroups also confirmed in this study were L, T, and Q. G1 was detected for the first time in the Croatian population. CONCLUSION: We obtained a new insight into the differences between examined subpopulations of Croatia and their possible (dis)similarities with neighboring and distant populations.

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

AIM: To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuski, Bosnia and Herzegovina. METHODS: Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. RESULTS: A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. CONCLUSION: DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons' relatives and collect referent samples from them.

Implementing Whole Genome Sequencing (WGS) in Clinical Practice: Advantages, Challenges, and Future Perspectives.

The integration of whole genome sequencing (WGS) into all aspects of modern medicine represents the next step in the evolution of healthcare. Using this technology, scientists and physicians can observe the entire human genome comprehensively, generating a plethora of new sequencing data. Modern computational analysis entails advanced algorithms for variant detection, as well as complex models for classification. Data science and machine learning play a crucial role in the processing and interpretation of results, using enormous databases and statistics to discover new and support current genotype-phenotype correlations. In clinical practice, this technology has greatly enabled the development of personalized medicine, approaching each patient individually and in accordance with their genetic and biochemical profile. The most propulsive areas include rare disease genomics, oncogenomics, pharmacogenomics, neonatal screening, and infectious disease genomics. Another crucial application of WGS lies in the field of multi-omics, working towards the complete integration of human biomolecular data. Further technological development of sequencing technologies has led to the birth of third and fourth-generation sequencing, which include long-read sequencing, single-cell genomics, and nanopore sequencing. These technologies, alongside their continued implementation into medical research and practice, show great promise for the future of the field of medicine.

Genetic polymorphisms of 15 STR loci in the population of the island of Cres (Croatia).

BACKGROUND: The population of the island of Cres presents one of the few persisting Eastern Adriatic isolates and is thereby suitable for human population differentiation analyses. AIM: The aim of this study was to analyse the genetic structure of the island of Cres with respect to its eight sub-populations and to compare the genetic variation of the island of Cres with other Eastern Adriatic islands and the Croatian mainland. SUBJECTS AND METHODS: Fifteen AmpFlSTR identifiler loci were analysed in a sample group of 122 unrelated autochthonous individuals from the island of Cres, Croatia. RESULTS: Analysis of STR polymorphisms revealed genetic homogeneity among sub-populations of the island of Cres and small but significant levels of genetic heterogeneity among geographically distant Eastern Adriatic islands. CONCLUSION: Despite a considerable degree of genetic homogeneity among the studied Eastern Adriatic islands, small but significant differentiation between distant islands indicates geographic sub-structuring which follows the isolation by distance model. This study is supportive of the notion that STR markers are useful for genetic differentiation between larger and geographically more distant regions.

Haplogroup Prediction Using Y-Chromosomal Short Tandem Repeats in the General Population of Bosnia and Herzegovina.

Human Y-chromosomal haplogroups are an important tool used in population genetics and forensic genetics. A conventional method used for Y haplogroup assignment is based on a set of Y-single nucleotide polymorphism (SNP) markers deployed, which exploits the low mutation rate nature of these markers. Y chromosome haplogroups can be successfully predicted from Y-short tandem repeat (STR) markers using different software packages, and this method gained much attention recently due to its labor-, time-, and cost-effectiveness. The present study was based on the analysis of a total of 480 adult male buccal swab samples collected from different regions of Bosnia and Herzegovina. Y haplogroup prediction was performed using Whit Athey's Haplogroup Predictor, based on haplotype data on 23 Y-STR markers contained within the PowerPlex® Y23 kit. The results revealed the existence of 14 different haplogroups, with I2a, R1a, and E1b1b being the most prevalent with frequencies of 43.13, 14.79, and 14.58%, respectively. Compared to the previously published studies on Bosnian-Herzegovinian population based on Y-SNP and Y-STR data, this study represents an upgrade of molecular genetic data with a significantly larger number of samples, thus offering more accurate results and higher probability of detecting rare haplogroups.

Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach.

AIM: To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. METHODS: Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Skofja Loka area (Lovrenska Grapa and Zolsce) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpF/STR MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. RESULTS: Following the Y-STR analysis, 1 of the "weak matches" previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Zolosce, we were able to obtain 6 useful DNA profiles. CONCLUSION: The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and mini-STR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II.

Forensic efficiency parameters for the 15 STR loci in the population of the island of Cres (Croatia).

Forensic parameters based on 15 AmpFISTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA) were evaluated in the sample of 122 unrelated, autochthonous, adult individuals from the Island of Cres (Croatia). PCR amplification was performed with the AmpFISTR Identifiler PCR Amplification Kit and the amplified products were separated and detected using the ABI 3130 DNA genetic analyzer. The agreement with Hardy Weinberg Equilibrium (HWE) was confirmed for all loci (p > 0.05). The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 tested STR loci were 0.99999999999999997988728679 and 0.999997397, respectively. According to the presented data, D18S51 proved to be the most informative marker followed by markers D2S1338 and D21S11. Interpopulation comparisons in allele frequencies with other East Adriatic Islands revealed significant differences for all analyzed population pairs ranging from 4 loci (Cres vs. Hvar) to 1 locus (Cres vs. Krk). Furthermore, allele frequencies comparisons of Cres and Croatian mainland revealed the lack of statistically significant differences at all studied loci. The results of the current study indicate that the examined fifteen STR loci are useful genetic markers for individual identification and paternity testing in Croatian population from the Island of Cres.

Allele frequencies for 15 short tandem repeat loci in representative sample of Croatian population.

AIM: To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of Croatian population. METHODS: A total of 195 unrelated Caucasian individuals born in Croatia, from 14 counties and the City of Zagreb, were sampled for the analysis. All the tested individuals were voluntary donors. Buccal swab was used as the DNA source. AmpFlSTR Identifiler was applied to simultaneously amplify 15 STR loci. Total reaction volume was 12.5 microL. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler. Electrophoresis of the amplification products was preformed on an ABI PRISM 3130 Genetic Analyzer. After PCR amplification and separation by electrophoresis, raw data were compiled, analyzed, and numerical allele designations of the profiles were obtained. Deviation from Hardy-Weinberg equilibrium, observed and expected heterozygosity, power of discrimination, and power of exclusion were calculated. Bonferroni's correction was used before each comparative analysis. RESULTS: We compared Croatian data with those obtained from geographically neighboring European populations. The significant difference (at P<0.01) in allele frequencies was recorded only between Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci. CONCLUSION: Obtained population data concurred with the expected "STR data frame" for this part of Europe.

DNA identification of skeletal remains from the World War II mass graves uncovered in Slovenia.

AIM: To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Skofja Loka, Slovenia. METHODS: The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. Quantifiler Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. RESULTS: Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. CONCLUSION: Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project.

Pathobiology of HER 2 positive small invasive (T1a,b) breast cancer: "A wolf in sheep's clothing".

Small invasive breast cancers (cancers with maximum diameter <1cm, T1a,b) become more prevalent form of breast cancer as a result of the introduction of breast cancer mammographic screening programs. Although associated with an excellent prognosis, T1a,b breast cancers are heterogeneous group of tumors with prognostically unfavorable subset of cases, primarily those with axillary lymph node metastases. To determine if the HER2 overexpression is associated with the prognostically unfavorable traditional clinicopathological features in this group of breast cancers, clinicopathological features (age, tumor size, histological type, histological grade, nodal status, hormone receptor status, proliferation index, lymphovascular invasion, ploidy) of 38 HER2 positive T1a,b cancers were compared with those of the control group consisting of 315 HER2 negative T1a,b cancers. The comparison of clinicopathological features was made using χ2 and t-test. HER2 positive T1a,b breast cancers were significantly associated with higher tumor grades (p<0.001), negative hormone receptors (p=0.008), presence of lymphovascular invasion (p=0.025), high proliferation index (p<0.001), and abnormal DNA content (p=0.04). We also noticed the higher frequency of lymph node positive cases in the HER2 positive group of cancers (p=0.05). There were no differences in age, tumor size and histological type between investigated groups. Our group of HER2 positive T1a,b breast cancers was associated with many unfavorable traditional prognostic factors, demonstrating that this subtype of early breast cancer has an aggressive biological phenotype which may have potential benefit from adjuvant chemo and immunotherapy.

Genetic structure and admixture between Bayash Roma from northwestern Croatia and general Croatian population: evidence from Bayesian clustering analysis.

The European Roma represent a transnational mosaic of minority population groups with different migration histories and contrasting experiences in their interactions with majority populations across the European continent. Although historical genetic contributions of European lineages to the Roma pool were investigated before, the extent of contemporary genetic admixture between Bayash Roma and non-Romani majority population remains elusive. The aim of this study was to assess the genetic structure of the Bayash Roma population from northwestern Croatia and the general Croatian population and to investigate the extent of admixture between them. A set of genetic data from two original studies (100 Bayash Roma from northwestern Croatia and 195 individuals from the general Croatian population) was analyzed by Bayesian clustering implemented in STRUCTURE software. By re-analyzing published data we intended to focus for the first time on genetic differentiation and structure and in doing so we clearly pointed to the importance of considering social phenomena in understanding genetic structuring. Our results demonstrated that two population clusters best explain the genetic structure, which is consistent with social exclusion of Roma and the demographic history of Bayash Roma who have settled in NW Croatia only about 150 years ago and mostly applied rules of endogamy. The presence of admixture was revealed, while the percentage of non-Croatian individuals in general Croatian population was approximately twofold higher than the percentage of non-Romani individuals in Roma population corroborating the presence of ethnomimicry in Roma.

Flow cytometric DNA hypertetraploidy tends to be more frequent in male than in female breast cancers.

The aim of the study was to explore possible differences in DNA flow cytometric characteristics, particularly differences in distribution of DNA indices of aneuploid clones, between male and female breast cancers. We retrospectively analyzed 31 male breast cancers. Clinicopathological and DNA flow cytometric characteristics of male breast cancers (patient age, tumor size, histological type, histological grade, axillary lymph node status, hormone receptor expression, ploidy, and S-phase fraction) were compared with that of the control group of matched female breast cancers. Hormone receptors and HER-2/neu were investigated immunohistochemically with additional chromogenic in situ hybridization (CISH) analysis of HER-2/neu 2+ cases. Ploidy and S-phase fraction were determined by DNA flow cytometry. Comparison with clinicopathological features was made using χ (2) and t test. Aneuploidy was found in 78% of the cases, with the predomination of hypotetraploid clones (39%), followed by tetraploid (23%) and hypertetraploid clones (16%). We found higher frequency of hypertetraploidy in male breast cancers (16 and 6%, respectively) than in the control group of matched female breast cancers. Clinicopathological features of hypertetraploid male breast cancers did not differ from that of non-hypertetraploid cancers. Higher frequency of hypertetraploidy among male breast cancers might indicate different cytogenetical evolutionary pathway between male and female breast cancer.

A comparative analysis of the effectiveness of cytogenetic and molecular genetic methods in the detection of Down syndrome.

The goal of this study was to examine the effectiveness of 6 STR markers application (D21S1435, D21S11, D21S1270, D21S1411, D21S226 and IFNAR) in molecular genetic diagnostics of Down syndrome (DS) and to compare it with cytogenetic method. Testing was performed on 73 children, with the previously cytogenetically confirmed Down syndrome. DNA isolated from the buccal swab was used. Previously mentioned loci located on chromosome 21 were simultaneously amplified using quantitative fluorescence PCR (QF PCR). Using this method, 60 previously cytogenetically diagnosed DS with standard type of trisomy 21 were confirmed. Furthermore, six of eight children with mosaic type of DS were detected. Two false negative results for mosaic type of DS were obtained. Finally, five children with the translocation type of Down syndrome were also confirmed with this molecular test. In conclusion, molecular genetic analysis of STR loci is fast, cheap and simple method that could be used in detection of DS. Regarding possible false results detected for certain number of mosaic types, cytogenetic analysis should be used as a confirmatory test.

Near-diploid hyperploidy in early breast cancer (T1a,b) is associated with higher risk of lymph node involvement.

Due to the worldwide implementation of the mammographic screening program early breast cancer (T1a,b) has become more prevalent form of breast cancer. Although T1a,b breast cancers are generally associated with excellent prognosis, some of them, particularly those with lymph node involvement, has unfavourable outcome. Searching for additional prognostic factors, we investigated DNA content of 163 T1a,b cancers measured by DNA flow cytometry, and correlated it with regional lymph node status. T1a,b cancers were divided into four ploidy classes based on their DNA index (DI): hypodiploid (DI < 0.95), diploid (DI 0.95-1.05), low-hyperploid (DI 1.06-1.3), and high-hyperploid (DI > 1.3). Diploid T1a,b cancers were associated with negative lymph node status (p = 0.003). Among aneuploid cancers only low-hyperploid tumors were associated with positive lymph node status (p = 0.03). The histopathological features of low-hyperploid group of T1a,b cancers did not differ from the other three ploidy groups of cancers, except for lower S-phase fraction of tumor cells in low-hyperploid group compared to high-hyperploid group (p = 0.01). Our data showed that near-diploid hyperploid T1a,b cancers are associated with higher risk of lymph node involvement despite similar clinicopathological features shared with other ploidy classes of T1a,b tumors.