Inherited and De Novo Variation in Lithuanian Genomes: Introduction to the Analysis of the Generational Shift.

Most genetic variants are rare and specific to the population, highlighting the importance of characterizing local population genetic diversity. Many countries have initiated population-based whole-genome sequencing (WGS) studies. Genomic variation within Lithuanian families are not available in the public databases. Here, we describe initial findings of a high-coverage (an average of 36.27×) whole genome sequencing for 25 trios of the Lithuanian population. Each genome on average carried approximately 4,701,473 (±28,255) variants, where 80.6% (3,787,626) were single nucleotide polymorphisms (SNPs), and the rest 19.4% were indels. An average of 12.45% was novel according to dbSNP (build 150). The WGS structural variation (SV) analysis identified on average 9133 (±85.10) SVs, of which 95.85% were novel. De novo single nucleotide variation (SNV) analysis identified 4417 variants, where 1.1% de novo SNVs were exonic, 43.9% intronic, 51.9% intergenic, and the rest 3.13% in UTR or downstream sequence. Three potential pathogenic de novo variants in the ZSWIM8, CDC42EP1, and RELA genes were identified. Our findings provide useful information on local human population genomic variation, especially for de novo variants, and will be a valuable resource for further genetic studies, and medical implications.

Genome-Wide Landscape of North-Eastern European Populations: A View from Lithuania.

There are still several unanswered questions regarding about ancient events in the Lithuanian population. The Lithuanians, as the subject of this study, are of great interest as they represent a partially isolated population maintaining an ancient genetic composition and show genetic uniqueness in European comparisons. To elucidate the genetic relationships between the Lithuania and North-Eastern European and West Siberian populations, we analyzed the population structure, effective population size, and recent positive selection from genome-wide single nucleotide polymorphism (SNP) data. We identified the close genetic proximity of Lithuanians to neighboring populations (Latvians, Estonians, Belarusians) and in part with West and South Slavs (Poles, Slovaks, and Slovenians), however, with particular genetic distinctiveness. The estimated long-term Ne values ranged from ~5900 in the Estonian population to ~2400 in the South Russian population. The divergence times between the Lithuanian and study populations ranged from 240 to 12,871 YBP. We also found evidence of selection in 24 regions, 21 of which have not been discovered in previous analyses of selection. Undoubtedly, the genetic diversity analysis of geographically specific regions may provide new insights into microevolutionary processes affecting local human populations.

The Effect of Prolonged Freezing and Holder Pasteurization on the Macronutrient and Bioactive Protein Compositions of Human Milk.

Introduction: Human donor milk is widely used to feed premature and sick newborns when the milk of their own mothers is insufficient. All treatment processes involving human milk affect its composition. The aim of this study was to assess changes in the macronutrients and bioactive protein (lactoferrin and lysozyme) content in human milk caused by freezing and Holder pasteurization. Materials and Methods: Milk samples were collected from 42 mothers 14-16 days after delivery. Each sample was divided into two parts and tested twice for macronutrient content, once upon being freshly collected and again after freezing at -40°C, thawing and Holder pasteurization. The lysozyme and lactoferrin concentrations were first determined in the unpasteurized thawed human milk after it was stored frozen at -80°C for up to 10 months and again after Holder pasteurization. The macronutrient concentrations were determined by midinfrared spectrophotometry, and enzyme-linked immunosorbent assay was used to measure the lysozyme and lactoferrin concentrations. Results: Freezing and Holder pasteurization had no significant effects on the macronutrient concentrations. The mean lactoferrin content before and after pasteurization was 2.5 ± 1.07 and 0.03 ± 0.03 mg/mL, respectively (p < 0.001), and the lysozyme content was 19.57 ± 20.11 and 12.62 ± 14.14 µg/mL, respectively (p = 0.007). Conclusions: Freezing and Holder pasteurization did not decrease the nutritional value of human milk but caused considerable loss of lactoferrin and lysozyme. New methods for treating human milk are needed that ensure the destruction of pathogenic microorganisms while retaining the biological and nutritional value of the milk. The Clinical Trial Registration number: NCT04382989.

Circadian changes in the composition of human milk macronutrients depending on pregnancy duration: a cross-sectional study.

BACKGROUND: The purpose of this study was to evaluate the circadian variation of human milk macronutrients and energy content depending upon pregnancy duration. METHODS: One hundred eighty fresh human milk samples from 45 mothers (27 of preterm and 18 of full-term newborns) were collected on a single day chosen between the 14th to 16th day after delivery. The samples were taken four times per day at 12 PM, 6 PM, 12 AM and 6 AM. Only lactating women, who could not breastfeed their hospitalized newborns and expressed milk by breast pump, were enrolled in the study. Human milk macronutrient composition and energy count were evaluated by mid-infrared spectrophotometry. RESULTS: Significant differences in macronutrient content were observed between 6 AM and 12 PM for mean protein content (t = - 4.62, df = 44, p < 0.001), for mean fat content (t = - 2.10, df = 44, p = 0.04) and for mean energy content (t = - 2.24, df = 44, p = 0.03); between 6 AM and 6 PM for mean protein content (t = - 2.41, df = 43, p = 0.02), for mean fat content (t = - 3.76, df = 43, p = 0.001) and for mean energy content (t = - 3.85, df = 43, p < 0.001); between 12 PM and 12 AM for mean protein content (Wilcoxon test V = 75.5, p = 0.001), for mean fat content (t = 2.50, df = 44, p = 0.02) and for mean energy content (t = 2.74, df = 44, p = 0.01); between 6 PM and 12 AM for mean protein content (V = 229, p = 0.02), for mean fat content (t = 4.39, df = 43, p < 0.001) and for mean energy content (t = - 4.57, df = 43, p < 0.001). The average content of carbohydrates did not change significantly during the 24 h. The samples of preterm newborns' mothers had more apparent diurnal fluctuations in macronutrient content. CONCLUSIONS: Our study revealed significant diurnal variations in protein and fat in human milk, and these circadian fluctuations were more apparent in the milk of mothers of preterm infants.

Inferring Effective Population Size and Divergence Time in the Lithuanian Population According to High-Density Genotyping Data.

The prehistory of the Lithuanian population and genetic relationship to other populations are poorly studied. Thus, the Lithuanian population, as an object of study, is interesting due to its partial isolation with genetic distinctiveness within the European context and with preserved ancient genetic composition. The main objects of this study was to infer demographic parameters, effective population size (Ne), and divergence time using high-density single nucleotide polymorphism (SNP) genotyping data generated with the Illumina HumanOmmiExpress-12v1.1 array in 295 individuals from the Lithuanian population and to compare our data with other populations from the Human Genome Cell Line Diversity Panel (HGDP-CEPH). We also aimed to reconstruct past events between the main ethnolinguistic regions-Aukstaitija and Zemaitija of Lithuania. Historically, these regions probably developed as two independent Baltic tribes. Our results of Ne in the Lithuanian population through time demonstrated a substantial reduction of Ne over the 150,000-25,000 years before present (YBP). The estimated long-term Ne of the Lithuanian population is quite low-it equals 5404, which likely is a consequence of the bottlenecks associated with the last glacial period of 25,000-12,000 YBP in Europe. The obtained divergence time estimates between the study populations are in agreement with recent studies. The reconstructed past events in Aukstaitija and Zemaitija showed significant differences between these two regions of Lithuania.

Analysis of Lithuanian CYP2D6 polymorphism and its relevance to psychiatric care of the local population.

BACKGROUND: CYP450 system gene CYP2D6 polymorphisms have been associated with an altered response to psychotropic drugs. While there exists interindividual and interethnic differences of clinical significance, there is no data concerning the Lithuanian population. AIMS: To determine the distribution of CYP2D6 alleles and predicted phenotype in the Lithuanian population, compare it to other Europeans and find the differences between patients with affective disorders and the healthy population. METHODS: Our study sample consisted of 179 subjects that included 104 healthy volunteers and 75 patients with clinical diagnosis of affective disorders according to ICD-10AM classification, treated in hospital settings. DNA samples were taken from the blood and alleles of the CYP2D6 gene were determined for each participant. Frequencies were compared to other Europeans. RESULTS: The frequency of the most common alleles *1 and *2 was 45.0% and 28.8% accordingly. Dysfunctional *5 (1 vs. 30, p < .002) allele was less frequent in Lithuania inhabitants than previously established in other Europeans. There were no polymorphisms of the CYP2D6 gene that could be associated with changes in drug metabolism in the patients. The functional CYP2D6 *2 allele was more prevalent in the control group, while the non functional CYP2D6 *4 allele was more prevalent in the patient group (p < .05 for both cases). CONCLUSION: The genetic makeup of Lithuanians was generally comparable to other Europeans, but fewer Lithuanians had non-functional *5 allele. More patients had non-functional alleles. Study findings contradict previous results from other countries, where CYP2D6 gene polymorphism was associated with treatment outcomes.

A comparative analysis of mathematical methods for homogeneity estimation of the Lithuanian population.

BACKGROUND: Population genetic structure is one of the most important population genetic parameters revealing its demographic features. The aim of this study was to evaluate the homogeneity of the  Lithuanian population on the  basis of the  genome-wide genotyping data. The comparative analysis of three methods - multidimensional scaling, principal components, and principal coordinates analysis - to visualize multidimensional genetics data was performed. The results of visualization (mapping images) are also presented. MATERIALS AND METHODS: The  data set consisted of 425 samples from six ethnolinguistic groups of the Lithuanian population. Genomic DNA was extracted from whole venous blood using either the phenol-chloroform extraction method or the automated DNA extraction platform TECAN Freedom EVO. Genotyping was performed at the Department of Human and Medical Genetics, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Lithuania, with the Illumina HumanOmniExpress-12 v1.1 and the Infinium OmniExpress-24. For the estimation of homogeneity of the Lithuanian population, PLINK data file was obtained using PLINK v1.07 program. The  Past3 software was used to visualize the genotype data with multidimensional scaling and principal coordinates methods. The  SmartPCA from EIGENSOFT 7.2.1 program was used in the principal component analysis to determine the population structure. CONCLUSIONS: Methods of multidimensional scaling, principal coordinate, and principal component for the genetic structure of the  Lithuanian population were investigated and compared. The principal coordinate and principal component methods can be used for genotyping data visualization, since any essential differences in the results obtained were not observed and compared to multidimensional scaling. The  Lithuanian population is homogenous whereas the points are strongly close when we use the principal coordinates or principal component methods.

Age, Sex, Body Mass Index, Education, and Social Support Influence Functional Results After Total Knee Arthroplasty.

INTRODUCTION: Total knee arthroplasty (TKA) is an effective treatment for knee osteoarthritis. Patient-reported outcome after TKA is influenced by multiple patient-related factors. The aim of this study was to prospectively evaluate preoperative patient-related factors and to compare the self-reported outcomes 1 year after TKA among groups differing by age, sex, body mass index (BMI), education, and social support level. METHODS: 314 patients, who underwent TKA in Vilnius Republican University Hospital between the end of 2012 and the middle of 2014, were included in a study. The preoperative and 12-month follow-up measurements were obtained using Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Short Form-12 (SF-12). Differences between patient groups according to gender, age, BMI, level of education, and level of social support were analyzed. RESULTS: At 12-month follow-up men demonstrated better results than women in WOMAC (P = .003) and SF-12 both domains (P < .05). Patients with a higher social support demonstrated higher scores in physical function according to SF-12 (P = .008). Better preoperative WOMAC and SF-12 scores were a predictor of better outcome 1 year after surgery. There was no difference in postoperative scores in different age, BMI, and education groups according to WOMAC and SF-12. CONCLUSION: There is no difference in self-reported functional outcome between patient groups differing in age, BMI, and education. Men and socially supported patients demonstrate better postoperative functional results 12 months after TKA. Better preoperative knee function and overall physical and mental function are predictors of better outcome 1 year after TKA. Age and obesity should not be limiting factors when considering who should receive this surgery.

Considering specific clinical features as evidence of pathogenic copy number variants.

Since the introduction of high-resolution microarray technologies, it has become apparent that structural chromosomal rearrangements can lead to a wide variety of clinical manifestations, including developmental delay/intellectual disability (DD/ID). It has been shown previously that the diagnostic yield of genome-wide array-based identification of submicroscopic alterations in patients with ID varies widely and depends on the patient selection criteria. More attempts have recently been made to define the phenotypic clues of pathogenic copy number variants (CNVs). The aim of this study was to investigate a well-phenotyped cohort of patients with DD/ID and determine whether certain clinical features may serve as indicators for pathogenic CNVs. A retrospective analysis was conducted for patients with DD/ID (n = 211) who were tested using genome-wide chromosomal microarray technologies and a review of the clinical data was performed. Pathogenic CNVs were detected in 29 patients. In comparison with individuals who had normal molecular karyotyping results (n = 182), malformations of the musculoskeletal system; congenital malformations of the CNS (particularly hydrocephalus and congenital malformations of the corpus callosum); minor anomalies of the eye, face, and neck subgroup (particularly downward-slanting palpebral fissures, minor anomalies of the ear, and micrognathia); brachydactyly; and umbilical hernia were more common in patients with chromosomal alterations. A multivariate logistic regression analysis allowed the identification of three independent pathogenic CNV predictors: congenital malformations of the corpus callosum, minor anomalies of the ear, and brachydactyly. Insights into the chromosomal phenotype may help to increase the diagnostic yield of microarray technologies and sharpen the distinction between chromosomal alterations and other conditions.

Heterogeneity of oral clefts in relation to associated congenital anomalies.

BACKGROUND AND OBJECTIVE: The first step in the search for the genetic basis of oral clefts should be the well-accepted classification and clinical data protocols, which are important in distinguishing separate phenotypic groups. The aim of this study was to compare the frequency of congenital malformations associated with oral clefts between the different groups of oral clefts. MATERIAL AND METHODS: The study population comprised 238 patients with oral clefts and one or more major congenital anomalies. All cases of oral clefts were subdivided into 2 groups: patients with the recognized conditions (n=97, 40.8%) and patients with the multiple congenital anomalies of unknown origin (n=141, 59.2%). The frequency of associated congenital anomalies was compared between the cleft palate (CP) and cleft lip and/or palate (CL/P) groups as well as between the cleft lip only (CL) and cleft lip with cleft palate (CLP) subgroups. RESULTS: A total of 420 anomalies associated with oral clefts were diagnosed in 141 patients with multiple congenital anomalies (2.98 anomalies per proband) with the highest incidence being in the CP group (3.5 anomalies per proband). Comparison of the CP and CL/P groups showed that some of associated congenital anomalies such as atresia and stenosis of the small intestine and micrognathia occurred significantly more often in the CP than CL/P group (2.1% vs. 0% and 3.5% vs. 1.1%; P<0.05). Meanwhile, comparison of the CL and CLP subgroups revealed accessory auricle, other specified anomalies of the ear, congenital anomalies of the circulatory system, and certain congenital musculoskeletal deformities of the spine to be more common in the CL than CLP group (5.1% and 0.5%, 11.9% and 5.1%, 3.4% and 0%, 3.4% and 0%, respectively; P<0.05). CONCLUSIONS: The highest incidence of associated congenital anomalies was in the CP group followed by the CL, CL/P, and CLP groups. Generally, the anomalies of the musculoskeletal system, cardiovascular system, and face including eye, ear, and neck were most common. The careful analysis of associated anomalies and cases of oral cleft subgroups with multiple congenital anomalies is helpful in identifying the etiologic entities and underscores the need for thorough evaluation and competent distinction of various types of oral clefts.