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Puberty onset is a developmental process influenced by genetic determinants, environment, and nutrition. Mutations and regulatory gene networks constitute the molecular basis for the genetic determinants of puberty onset. The emerging knowledge of these genetic determinants presents opportunities for innovation in the breeding of early pubertal cattle. This paper presents new data on hypothalamic gene expression related to puberty in Bos indicus (Brahman) in age- and weight-matched heifers. Six postpubertal heifers were compared with 6 prepubertal heifers using whole-genome RNA sequencing methodology for quantification of global gene expression in the hypothalamus. Five transcription factors (TF) with potential regulatory roles in the hypothalamus were identified in this experiment: E2F8, NFAT5, SIX5, ZBTB38, and ZNF605. These TF genes were significantly differentially expressed in the hypothalamus of postpubertal versus prepubertal heifers and were also identified as significant according to the applied regulatory impact factor metric (P < 0.05). Two of these 5 TF, ZBTB38 and ZNF605, were zinc fingers, belonging to a gene family previously reported to have a central regulatory role in mammalian puberty. The SIX5 gene belongs to the family of homologues of Drosophila sine oculis (SIX) genes implicated in transcriptional regulation of gonadotrope gene expression. Tumor-related genes such as E2F8 and NFAT5 are known to affect basic cellular processes that are relevant in both cancer and developmental processes. Mutations in NFAT5 were associated with puberty in humans. Mutations in these TF, together with other genetic determinants previously discovered, could be used in genomic selection to predict the genetic merit of cattle (i.e., the likelihood of the offspring presenting earlier than average puberty for Brahman). Knowledge of key mutations involved in genetic traits is an advantage for genomic prediction because it can increase its accuracy. [ABSTRACT FROM AUTHOR]

The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth. [ABSTRACT FROM AUTHOR]

During the last months, the number of reports on Holstein calves suffering from incurable idiopathic diarrhea dramatically increased. Affected calves showed severe hypocholesterolemia and mostly died within days up to a few months after birth. This new autosomal monogenic recessive inherited fat metabolism disorder, termed cholesterol deficiency (CD), is caused by a loss of function mutation of the bovine APOB gene. The objective of the present study was to investigate specific components of lipid metabolism in 6 homozygous for the APOB mutation (CDS) and 6 normal Holstein calves with different APOB genotypes. Independent of sex, CDS had significantly lower plasma concentrations of total cholesterol (TC), free cholesterol (FC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-lowdensity lipoprotein cholesterol (VLDL-C), triacylglycerides (TAG), and phospholipids (PL) compared with homozygous wild-type calves (P < 0.05). Furthermore, we studied the effect of the APOB genotype on cholesterol metabolism in adult Holstein breeding bulls of Swissgenetics. Among a total of 254 adult males, the homozygous mutant genotype was absent, 36 bulls were heterozygous carriers (CDC), and 218 bulls were homozygous wild-type (CDF). In CDC bulls, plasma concentrations of TC, FC, HDL-C, LDL-C, VLDL-C, TAG, and PL were lower compared with CDF bulls (P < 0.05). The ratios of FC:cholesteryl esters (CE) and FC:TC were higher in CDC bulls compared with CDF bulls, whereas the ratio of CE:TC was lower in CDC bulls compared with CDF bulls (P < 0.01). In conclusion, the CD-associated APOB mutation was shown to affect lipid metabolism in affected Holstein calves and adult breeding bulls. Besides cholesterol, the concentrations of PL, TAG, and lipoproteins also were distinctly reduced in homozygous and heterozygous carriers of the APOB mutation. Beyond malabsorption of dietary lipids, deleterious effects of apolipoprotein B deficiency on hepatic lipid metabolism, steroid biosynthesis, and cell membrane function can be expected, which may result in unspecific symptoms of reduced fertility, growth, and health. [ABSTRACT FROM AUTHOR]

Combining purebreed and crossbreed information is beneficial for genetic evaluation of some livestock species. Genetic evaluations can use relationships based on genomic information, relying on allele frequencies that are breed specific. Singlestep genomic BLUP (ssGBLUP) does not account for different allele frequencies, which could limit the genetic gain in crossbreed evaluations. In this study, we tested the performance of different breed-specific genomic relationship matrices (GB) in ssGBLUP for crossbreed evaluations; we also tested the importance of genotyping crossbred animals. Genotypes were available for purebreeds (AA and BB) and crossbreeds (F1) in simulated and real pig populations. The number of genotyped animals was, on average, 4,315 for the simulated population and 15,798 for the real population. Cross-validation was performed on 1,200 and 3,117 F1 animals in the simulated and real populations, respectively. Simulated scenarios were under no artificial selection, mass selection, or BLUP selection. Two genomic relationship matrices were constructed based on breed-specific allele frequencies: 1) GB1, a genomic relationship matrix centered by breed-specific allele frequencies, and 2) GB2, a genomic relationship matrix centered and scaled by breed-specific allele frequencies. All G (the across-breed genomic relationship matrix), GB1, and GB2 were also tuned to account for selective genotyping. Using breed-specific allele frequencies reduced the number of negative relationships between 2 purebreeds, pulling the average closer to 0, as in the pedigree-based relationship matrix. For simulated populations that included mass selection, genomic EBV (GEBV) in F1, when using GB1 and GB2, were, on average, 10% more accurate than G; however, after tuning to account for selective genotyping, G provided the same accuracy as for breed-specific genomic relationship matrices. For the real population, accuracies for litter size in F1 were 0.62 for G, GB1, and GB2, and tuning had no impact on accuracy, except for GB2, which was 1 percentage point less accurate. Accuracy of GEBV for number of stillborns in F1 was 0.5 for all tested genomic relationship matrices with no changes after tuning. We observed that genotyping F1 increased accuracies of GEBV for the same animals by up to 39% compared with having genotypes for only AA and BB. In crossbreed evaluations, accounting for breed-specific allele frequencies promoted changes in G that were not influential enough to improve accuracy of GEBV. Therefore, the best performance of ssGBLUP for crossbreed evaluations requires genotypes for pure- and crossbreeds and no breed-specific adjustments in the realized relationship matrix. [ABSTRACT FROM AUTHOR]

A SNP in a regulatory region of intron 3 within the porcine IGF2 gene (JGF2-G3072A) is associated with increased lean deposition and decreased fat deposition in pigs with paternal A alleles (APat) compared with pigs with paternal G alleles (GPat). However, data regarding fresh and processed meat quality characteristics of pigs with different alleles for this polymorphism are limited. A single heterozygote (AG) boar was bred to homozygous (AA) commercial Yorkshire-cross sows producing F1 barrows and gilts with either GPat or APat. Two farrowing groups of barrows and gilts were group housed, provided ad libitum access to a diet that met or exceeded NRC nutrient recommendations throughout production, and slaughtered at 176 d (±4 d) of age. Fresh LM quality and estimated percent fat-free lean measurements were taken on the left side of carcasses, while carcass cutouts were completed with right sides. Fresh belly and bacon processing traits were characterized for only block 1 pigs. Pig was treated as the experimental unit for all analyses. Ending live weight and HCW were not affected by IGF2 allele; however, 10th rib backfat thickness was 0.41 cm less (P = 0.01), loin eye area was 4.0 cm² greater (P = 0.01), and predicted fat-free lean was over 2 percentage units greater (P <0.01) in APat pigs compared with GPat pigs. Furthermore, boneless lean cuts from the shoulder, loin, and ham were heavier (P < 0.05) in APat pigs compared with GPat pigs. Minolta L* value was 2.36 units greater (P = 0.03) but cooking loss was 1.82(9839658256) percentage units greater (P <0.01) in APat pigs compared with GPat pigs. Additionally, despite reductions in subcutaneous fat, extractable intramuscular lipid from the LM was 0.64 percentage units greater (P = 0.02) in APat pigs compared with GPat pigs. Bellies were 7.17 mm thinner (P = 0.01), had 7.27 cm less flop distance (P = 0.05), and tended to have 1.34 units greater iodine value (P = 0.09) in APat pigs compared with GPat pigs. While not statistically different (P = 0.30), the magnitude of difference in slicing yield as a percentage of green weight was 1.57 percentage units between bellies from APat pigs (85.83%) and bellies from GPat pigs (87.40%). Pigs with GPat had superior belly quality that may positively impact commercial bacon production. However, pigs with APat yielded a greater amount of lean product at the expense of producing lighter LM color and increased cooking loss. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance in breeding animals and increased respiratory problems in growing animals, which result in significant economic losses in the swine industry. Vaccination has generally not been effective in the prevention of PRRS, partially because of the rapid mutation rate and evolution of the virus. The objective of the current study was to discover the genetic basis of host resistance or susceptibility to the PRRS virus through a genome-wide association study using data from the PRRS Host Genetics Consortium PRRS-CAP project. Three groups of approximately 190 commercial crossbred pigs from 1 breeding company were infected with PRRS virus between 18 and 28 d of age. Blood samples and BW were collected up to 42 d post infection (DPI). Pigs were genotyped with the Illumina Porcine 60k Beadchip. Whole-genome analysis focused on viremia at each day blood was collected and BW gains from 0 to 21 DPI (WG21) or 42 DPI (WG42). Viral load (VL) was quantified as area under the curve from 0 to 21 DPI. Heritabilities for WG42 and VL were moderate at 0.30 and litter accounted for an additional 14% of phenotypic variation. Genomic regions associated with VL were found on chromosomes 4 and X and on 1,4, 7, and 17 for WG42. The 1-Mb region identified on chromosome 4 influenced both WG and VL, exhibited strong linkage disequilibrium, and explained 15.7% of the genetic variance for VL and 11.2% for WG42. Despite a genetic correlation of-0.46 between VL and WG42, genomic EBV for this region were favorably and nearly perfectly correlated. The favorable allele for the most significant SNP in this region had a frequency of 0.16 and estimated allele substitution effects were significant (P < 0.01) for each group when the SNP was fitted as a fixed covariate in a model that included random polygenic effects with overall estimates of-4.1 units for VL (phenotypic SD = 6.9) and 2.0 kg (phenotypic SD = 3 kg) for WG42. Candidate genes in this region on SSC4 include the interferon induced guanylate-binding protein gene family. In conclusion, host response to experimental PRRS virus challenge has a strong genetic component, and a QTL on chromosome 4 explains a substantial proportion of the genetic variance in the studied population. These results could have a major impact in the swine industry by enabling marker-assisted selection to reduce the impact of PRRS but need to be validated in additional populations. [ABSTRACT FROM AUTHOR]

The Small Tailed Han is a prolific local sheep breed in China. The bone morphogenetic protein receptor IB (BMPR-IB) gene, which affects the fecundity of Booroola Merino sheep, and the bone morphogenetic protein 15 (BMP-15) gene, which affects the fecundity of Inverdale, Hanna, Belclare, Cambridge, and Lacaune sheep, were studied as candidate genes associated with the prolificacy of Small Tailed Han sheep. Single nucleotide polymorphisms of BMPR-IB and BMP-15 genes were detected in Small Tailed Han ewes (n = 188) by PCR-RFLP. The combined effect of the 2 genes on the prolificacy of Small Tailed Han sheep was studied. The results indicated that the same FecB mutation (Q249R) occurred in the BMPR-IB gene in Small Tailed Han ewes as found in Booroola Merino ewes. The Small Tailed Han ewes with genotypes FecBB/FecBB and FecBB/FecB+ had 1.40 (P < 0.01) and 1.11 (P < 0.01) more lambs, respectively, than those with genotype FecB+/FecB+. The same FecXG mutation (Q239Ter) of the BMP-15 gene was found in Small Tailed Han ewes as in Belclare and Cambridge ewes. The Small Tailed Han ewes with the heterozygous mutant FecXG/FecX+ had 0.55 (P < 0.01) more lambs than those with the wild-type FecX+/FecX+. The Small Tailed Han ewes carrying mutations in both BMPR-IB and BMP-15 genes had greater litter size than those with either mutation alone. In view of our results, marker-assisted selection using both BMPR-IB and BMP-15 genes is warranted to increase litter size in sheep and will be of considerable economic value to sheep producers. [ABSTRACT FROM AUTHOR]

Bovine syndactyly is an autosomal monogenic recessive trait associated with multiple mutations in the LRP4 gene encoding low-density lipoprotein receptorrelated protein 4. This protein is a specific facilitator of sclerostin-mediated inhibition of both Wnt1/β-catenin signaling and osteocyte-secreted bone formation. It is assumed that sclerostin induces a decrease in extracellular pH and promotes the release of calcium ions from mineralized substrates. Therefore, mutations in the LRP4 gene might be associated with changes in the elemental status. The aim of this research was to evaluate the elemental composition of serum in cattle carrying multiple SNP in the LRP4 gene (16 trial animals) compared to animals without such mutations (62 control animals). DNA samples from 78 Black Pied cattle were sequenced with a TruSeq Bovine Parentage Sequencing Panel (Illumina, USA) on a MiSeq Illumina platform following by SNP analysis. The elemental composition of the serum was determined using ICP-AES and ICP-MS. The levels of Ca and P, crucial bone elements, were higher in trial cattle than in control group by 12.9% (P = 0.004) and 22.5% (P = 0.008), respectively. The level of V affecting the uniform distribution of calcium salts in bones, was higher in trial group than in control one by 34.5% (P = 0.001). The concentrations of Mn and Mg were higher in trial group than in control one by 23.6% (P = 0.026) and 10.0% (P = 0.044), respectively. The Sr level in trial cattle was higher by 18.8% (P = 0.04) that may indicate the rickets-like state. A similar trend was observed for toxic metals As and Li. This study demonstrates the association of the LRP4 multiple SNP and the changes in cattle elemental status. Moreover, such heterozygous animals have a higher risk of mineral metabolism disorders. This research was supported by The Ministry of Science and Higher Education of the Russian Federation (Project No. 0526-2019-0001). [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the relative effects of the porcine IGF2 mutation (IGF2-G3072A) and a novel, gene-editing derived myostatin loss-of-function (LOF) mutation on carcass traits and meat quality in pigs. The hypothesis was that both the IGF2 paternal A allele (Apat) and myostatin LOF mutation would increase muscle growth in pigs, but may potentially reduce loin and belly quality. Pigs were either IGF2 paternal G allele (Gpat) and wild-type (WT) for myostatin (n=13), Gpat and heterozygous (HET) for myostatin (n=10), Apat WT (n=11) or Apat HET (n=10). Pigs were raised in mixed sex pens of 10-15 pigs per pen, given free access to a diet that met or exceeded NRC nutrient requirements, and slaughtered at 175 d (±5 d) of age. Loin eye area and back fat depth was measured. Loin and belly quality was measured on the left side of the carcass. Warner-Bratzler shear force (WBSF) was used to determine tenderness of loin chops d14 postmortem. Data were analyzed using the MIXED procedure of SAS with a model including the effects of sex, IGF2, myostatin, and all interactions; means were separated using the pdiff option and considered different when P≤0.05. Ending live weight and hot carcass weight were similar among all genotypes (P≥0.05). Loin eye area was greater (P≤0.05) in HET pigs regardless of IGF2 genotype compared with WT pigs, and tended (P=0.10) to be greater in IGF2 Apat WT pigs compared with Gpat WT pigs. Back fat depth was similar (P≥0.05) among all genotypes. Longissimus dorsi (LD) muscle weighed less (P≤0.05) in WT Gpat pigs in comparison with all other genotypes, and was larger (P≤0.05) in Apat HET pigs compared with Apat WT pigs. Loins from myostatin HET pigs were paler than those from WT pigs, regardless of IGF2 genotype, as evidenced by an increased L* value of approximately 5 units (P< 0.01). However, L* of IGF2 Apat (52.1) and Gpat (53.9) pigs were similar (P≥0.05). Tenderness (WBSF) was similar (P≥0.05) among all genotypes. Bellies from IGF2 Gpat pigs were firmer as indicated by an increased (P<0.05) flop distance compared with all other genotypes. These data suggest that the partial loss of myostatin (HET) increased lean muscle deposition in pigs but resulted in poorer loin color. The IGF2 Apat mutation also tended to increase lean muscle deposition but did not negatively impact loin color. However, both mutations resulted in poorer belly firmness. The objective of this study was to determine the relative effects of the porcine IGF2 mutation (IGF2-G3072A) and a novel, gene-editing derived myostatin loss-of-function (LOF) mutation on carcass traits and meat quality in pigs. The hypothesis was that both the IGF2 paternal A allele (Apat) and myostatin LOF mutation would increase muscle growth in pigs, but may potentially reduce loin and belly quality. Pigs were either IGF2 paternal G allele (Gpat) and wild-type (WT) for myostatin (n=13), Gpat and heterozygous (HET) for myostatin (n=10), Apat WT (n=11) or Apat HET (n=10). Pigs were raised in mixed sex pens of 10-15 pigs per pen, given free access to a diet that met or exceeded NRC nutrient requirements, and slaughtered at 175 d (±5 d) of age. Loin eye area and back fat depth was measured. Loin and belly quality was measured on the left side of the carcass. Warner-Bratzler shear force (WBSF) was used to determine tenderness of loin chops d14 postmortem. Data were analyzed using the MIXED procedure of SAS with a model including the effects of sex, IGF2, myostatin, and all interactions; means were separated using the pdiff option and considered different when P≤0.05. Ending live weight and hot carcass weight were similar among all genotypes (P≥0.05). Loin eye area was greater (P≤0.05) in HET pigs regardless of IGF2 genotype compared with WT pigs, and tended (P=0.10) to be greater in IGF2 Apat WT pigs compared with Gpat WT pigs. Back fat depth was similar (P≥0.05) among all genotypes. Longissimus dorsi (LD) muscle weighed less (P≤0.05) in WT Gpat pigs in comparison with all other genotypes, and was larger (P≤0.05) in Apat HET pigs compared with Apat WT pigs. Loins from myostatin HET pigs were paler than those from WT pigs, regardless of IGF2 genotype, as evidenced by an increased L* value of approximately 5 units (P< 0.01). However, L* of IGF2 Apat (52.1) and Gpat (53.9) pigs were similar (P≥0.05). Tenderness (WBSF) was similar (P≥0.05) among all genotypes. Bellies from IGF2 Gpat pigs were firmer as indicated by an increased (P<0.05) flop distance compared with all other genotypes. These data suggest that the partial loss of myostatin (HET) increased lean muscle deposition in pigs but resulted in poorer loin color. The IGF2 Apat mutation also tended to increase lean muscle deposition but did not negatively impact loin color. However, both mutations resulted in poorer belly firmness. [ABSTRACT FROM AUTHOR]

Optimizes polyacrylamide gel electrophoresis (PAGE) conditions for detecting the ryanodine receptor (RYR1) gene mutation in pigs as the adequate mobility shift. Collection of data from eight breeds of pigs; Diagnosis of polymerase chain reaction products(PCR); Incubation of PCR products.

Transmission ratio distortion (TRD) is the deviation from the expected Mendelian inheritance of alleles from heterozygous parents. This phenomenon has been reported in a broad range of organisms and can be caused by various biological mechanisms including meiotic drive, embryo or fetal failure, germline selection, gametic competition, imprint resetting error and differential survival of offspring. Within this context, we investigated TRD in cattle aiming to identify genomic regions showing altered deviations in segregation that could contain potential causal mutations directly affecting economically important traits, such as reproduction. A Bayesian approach was implemented in order to uncover TRD across the whole genome using SNP-by-SNP and haplotypes of 2-SNP sliding windows using 79,238 genotyped trios of Holstein. From a total of 44,369 autosomal SNP, 942 were significantly detected with TRD that exceeded a Bayes factor (BF) ≥ 10 (strong evidence) and 408 with BF ≥ 100 (decisive evidence) from SNP-by-SNP analyses. The number of SNP with parent-unspecific TRD were 270 whereas 393 and 271 SNP were detected as either sire- or dam-TRD, respectively. By using the approximate empirical null distribution of TRD, results potentially generated by chance were discarded, resulting in 555, 307 and 92 SNP significantly detected with a probability of error of 5%, 1% and 0.1%, respectively. From the SNP-by-SNP approach, regions with moderate-to-high |TRD| (≥ 0.15) were less polymorphic (minor allele frequency, MAF < 0.027), providing evidence of TRD selection and unravelling rare variants as candidate lethal alleles. In contrast, slightly more polymorphic regions with moderate-to-high |TRD| were identified by the haplotype approach (MAF ~ 0.045). The number of regions identified by haplotype approach after correction (at 0.1% probability of error) were 1,047, 421 and 683 for overall, sire- and dam-TRD, respectively. The preliminary functional analysis of detected regions with TRD identified positional genes associated with regulation of embryonic development. Moreover, the identification of alleles with TRD in chromosomal regions around haplotypes well known to be associated with recessive disorders in Holstein or affecting fertility traits in other cattle breeds highlighted important biological implications of TRD. In conclusion, the prevalence of TRD was extended across the whole genome and an in-depth deeper study of these candidate regions and alleles will be further investigated to better understand this phenomenon in cattle. [ABSTRACT FROM AUTHOR]

LoF (loss-of-function) mutations causing hereditary abnormalities and embryonic mortality are one of the reasons for a decline in fertility of cows. Currently, more than fifteen LoF mutations are known in Holsteins. In our work, we studied the frequency of LoF mutations in FANCI, APAF1, SMC2, GART, and APOB genes that are associated with fertility haplotypes HH0, HH1, HH3, HH4, and HCD in the Russian Holstein cattle population. We analyzed the estimated breeding values (EBVs) for daughters' milk production and reproduction traits in sires carrying LoF mutations compared to non-carrier sires using the BLUE approach. Genotyping LoF mutations was performed by PCR, PCR-RFLP or allele-specific PCR. In total, 1,521, 636, 880, 690, and 574 bulls and 896, 630, 773, 482, and 727 cows were genotyped for LoF mutations in FANCI, APAF1, SMC2, GART, and APOB genes, respectively. The ratio of carriers among bulls and cows was, respectively, 2.89 and 4.13% for FANCI, 2.04 and 1.83% for APAF1, 1.14 and 2.98% for SMC2, 1.30 and 1.04% for GART, and 5.57 and 2.06% for APOB. Generally, the sires carrying LoF mutations had higher EBVs for milk production traits. The greatest effect was observed for SMC2 genotype: +236 kg for 305-days milk yield, 9.0 kg for fat yield, and 7.6 kg for protein yield. The bulls carrying LoF mutations in the SMC2 and GART genes were characterized by the higher number semen doses per insemination (0.02 to 0.24 units) compared with the noncarrier bulls. The higher EBVs for the number of days open (from 12.6 (P < 0.001) to 7.2 days (P < 0.05)) and calving interval (from 9.0 (P < 0.001) to 10.5 days (P < 0.01)) were observed in bulls, which were carriers of LoF mutations in SMC2 and GART genes. The group of sires with the mutation in the FANCI gene had the smallest length of calving interval (-6.0 days, P < 0,001). Analysis of the best linear unbiased estimates showed that for insemination of offspring from bull carriers of HH3 haplotype spent +0.026 units semen than for bulls with other mutations. The longest interval from calving to the first insemination and days open by +1.9 to 3.9 days were daughters of bulls with the GART gene (HH4). Given the dissemination of LoF mutations among the breeding sires and cows, with the aim of reducing genetically caused embryonic losses, matings of carriers bulls with mutations to cows whose fathers are hidden carriers of LoF mutations in the FANCI, APAF1, SMC2, GART, and APOB gene must be excluded. [ABSTRACT FROM AUTHOR]