Molecular Karyotyping Research Articles

31. CHROMOSOMAL MOSAICISM AS A RISK FACTOR FOR INCORRECT RESULTS OF PGT-A

Introduction Mosaicism may cause discordance between the result of PGT-A and the karyotype of cells outside the biopsy area. How likely is the risk of incorrect results of PGT-A due to chromosomal mosaicism? The aim of present study was comparison of molecular karyotypes of trophectoderm cells and inner cell mass. The study presents the results of re-analysis of the molecular karyotypes of 10 embryos by the comparative genomic hybridization on microarrays (aCGH). Material & methods Initially, the trophectoderm biopsy was performed for all 10 embryos for PGT-A using the aCGH analysis (24sure, Illumina). All 10 trophectoderm samples were found to be aneuploid. Corresponding embryos were not recommended for transfer and were included in this study per patients’ informed consent. All embryos were divided into 2 parts: trophectoderm (TE) and the inner cell mass (ICM). Each part was analyzed separately. For 2 embryos blastomeres (B) not included in the blastocyst were also available (for embryos №2 and №3). Re-analysis was performed using the aCGH analysis (24sure, Illumina). Results Molecular karyotypes of TE cells and ICM 7 of 10 embryos coincided with PGT-A results. Molecular karyotypes of 3 samples demonstrated incomplete concordance with the PGT-A results. We detected mosaic form of aneuploidies of sex chromosomes in the inner cell mass of embryo №10. PGT-A result of this sample contains information about whole aneuploidy of sex chromosomes. Mosaic form of reciprocal aneuploidy was observed in the ICM of embryo №4. Additional monosomy of chromosome 16 was detected in TE of embryo №8. This aneuploidy was not detected in the ICM and in the trophectoderm biopsy sample for PGT-A. No preferential localization of abnormal cells in trophectoderm or inner cell mass was observed. The study has limitations: a) the complete separation cells one by one and TE from ICM at blastocyst stage is impossible; b) there is a risk of false determination of the localization of aneuploidy in the embryo and the risk of inability to detect aneuploidy in the case when the sample contains reciprocal aneuploidy. Conclusions Our results show the coincidence of the molecular karyotypes of different embryo's cells was detected for most samples. In cases of detection of mosaic aneuploidy in ICM, cells with the whole aneuploidy and the normal molecular karyotype were also present in TE. None of the 10 embryos was suitable for transfer per any sample results. Further research is required with increasing sample size. Mosaicism may cause discordance between the result of PGT-A and the karyotype of cells outside the biopsy area. How likely is the risk of incorrect results of PGT-A due to chromosomal mosaicism? The aim of present study was comparison of molecular karyotypes of trophectoderm cells and inner cell mass. The study presents the results of re-analysis of the molecular karyotypes of 10 embryos by the comparative genomic hybridization on microarrays (aCGH). Initially, the trophectoderm biopsy was performed for all 10 embryos for PGT-A using the aCGH analysis (24sure, Illumina). All 10 trophectoderm samples were found to be aneuploid. Corresponding embryos were not recommended for transfer and were included in this study per patients’ informed consent. All embryos were divided into 2 parts: trophectoderm (TE) and the inner cell mass (ICM). Each part was analyzed separately. For 2 embryos blastomeres (B) not included in the blastocyst were also available (for embryos №2 and №3). Re-analysis was performed using the aCGH analysis (24sure, Illumina). Molecular karyotypes of TE cells and ICM 7 of 10 embryos coincided with PGT-A results. Molecular karyotypes of 3 samples demonstrated incomplete concordance with the PGT-A results. We detected mosaic form of aneuploidies of sex chromosomes in the inner cell mass of embryo №10. PGT-A result of this sample contains information about whole aneuploidy of sex chromosomes. Mosaic form of reciprocal aneuploidy was observed in the ICM of embryo №4. Additional monosomy of chromosome 16 was detected in TE of embryo №8. This aneuploidy was not detected in the ICM and in the trophectoderm biopsy sample for PGT-A. No preferential localization of abnormal cells in trophectoderm or inner cell mass was observed. The study has limitations: a) the complete separation cells one by one and TE from ICM at blastocyst stage is impossible; b) there is a risk of false determination of the localization of aneuploidy in the embryo and the risk of inability to detect aneuploidy in the case when the sample contains reciprocal aneuploidy. Our results show the coincidence of the molecular karyotypes of different embryo's cells was detected for most samples. In cases of detection of mosaic aneuploidy in ICM, cells with the whole aneuploidy and the normal molecular karyotype were also present in TE. None of the 10 embryos was suitable for transfer per any sample results. Further research is required with increasing sample size.

39. CLINICAL INTERPRETATION OF NON-STANDARD PGT-A RESULTS

Introduction Preimplantation genetic testing for chromosome anomalies has been used to improve reproductive outcomes for nearly 20 years. But in some cases, the effectiveness of Preimplantation genetic testing for aneuploidies (PGT-A) may be reduced. Sources of errors during PGT-A are low sensitivity or specificity of either method or interpretation. The threshold value that separates normal result from pathological is not fully formalized for current methods of PGT-A (aCGH, NGS). The decision on the status of the sample is taken by the interpreter (the expert). Can preimplantation genetic testing for aneuploidies reduce IVF efficiency? Yes, if euploid embryo is defined as abnormal or if abnormal embryo is recommended for transfer. To estimate how much the interpretation of the same PGT-A data may differ between the readers we designed the project «Many men, many minds». Materials & Methods We compared the experts’ interpretation with some arbitrary reference. The references are the clinical reports, created in Genetico Laboratory, based on the joined decision after discussion of program data between the clinical geneticist, laboratory geneticist and biologist. Participants of the study were 10 readers, 2 of which are outside Russia. We included 50 samples analyzed by aCGH (Cytochip 24sure, Illumina) and 50 samples analyzed by NGS (Veriseq, Illumina) in our project. The samples for interpretation were not representative of routine samples of Genetico Laboratory (not typical samples were chosen for interpretation, but rather most hard to read ones). The experts received data which include chromosome profiles, QC metrics, and chromosome imbalances which automatically determined by the BlueFuse Multi program (Illumina). We asked experts to provide an answer containing molecular karyotype for each sample and clinical recommendations for embryo transfer. Results Interpretation of PGT-A results depends of expert's “school of thought” – at the same lab opinions tend to be more concordant. And interpretation of chromosome profiles by another expert can lead to a change in the clinical fate of the embryo. It is advisable to discuss PGT-A results between specialists before clinical report making. Interpretation of hard to read data of PGT-A is difficult and the role of the human factor very big. The “low specificity” of an expert may led to samples being recognized as aneuploid, although another expert using the same data will give an estimate of “norm” (or vice versa). Reanalysis of PGT-A data may lead to significant changes in clinical decisions regarding the fate of embryos. And after several months we tested if one interpreter could have two different opinions regarding the same sample. The participants of this additional experiment became 5 experts from initial list of participants. We asked them about the interpretation of 10 samples from the initial list. We did not reveal that these samples were same the experts interpreted several months before. We saw that the same expert may have different opinions on the same sample. Conclusions PGT-A allows to increase IVF efficiency, however, we have room for improvement. It is possible to work on improving the process of data interpretation.

Induced pluripotent stem cell line heterozygous for p.R501X mutation in filaggrin: KCLi003-A

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

Clinical use of SNP-microarrays for the detection of genome-wide changes in haematological malignancies.

Single nucleotide polymorphism (SNP) microarrays are commonly used for the clinical investigation of constitutional genomic disorders; however, their adoption for investigating somatic changes is being recognised. With increasing importance being placed on defining the cancer genome, a shift in technology is imperative at a clinical level. Microarray platforms have the potential to become frontline testing, replacing or complementing standard investigations such as FISH or karyotype. This 'molecular karyotype approach' exemplified by SNP-microarrays has distinct advantages in the investigation of several haematological malignancies. A growing body of literature, including guidelines, has shown support for the use of SNP-microarrays in the clinical laboratory to aid in a more accurate definition of the cancer genome. Understanding the benefits of this technology along with discussing the barriers to its implementation is necessary for the development and incorporation of SNP-microarrays in a clinical laboratory for the investigation of haematological malignancies.

Evaluation of two aneuploidy screening tests for chorionic villus samples: Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

The vast majority of first-trimester pregnancy losses are the consequence of numerical aberrations in fetal chromosomes, which may involve nearly all chromosomes. Although commercial probes for all chromosomes are available for multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) analyses, their use has rarely been reported for screening all 24 chromosomes for early fetal demise, especially by FISH. Here, we validated the ability of MLPA and FISH techniques as two low-cost aneuploidy screening methods for 24 chromosomes in 165 chorionic villus samples (CVSs). The results obtained by two methods were compared by the Chi-square test and the Kappa agreement test. Both methods gave conclusive results for all CVSs tested and showed highly consistent results (kappa = 0.890, p < 0.001). There was no statistically significant difference between the aneuploidy rate of the CVSs tested by the two methods (p = 0.180). Most of the samples showed fully concordant molecular karyotyping results (81.21%) between the two analytical methods, 10.91% had incompletely concordant results, and 7.88% had discordant results. The inconsistencies included segmental abnormalities, mosaicism, and polyploidy. Both assays used to screen 24 chromosomes were powerful techniques for detecting aneuploidy in CVSs. In terms of cost-effectiveness and diagnostic accuracy, the combination of subtelomeric (P036, P070) and centromeric (P181) MLPA assays is the better analytic strategy and follow-up analysis by FISH is recommended for MLPA-negative samples.

Diagnostic Yield of Molecular Karyotyping of Idiopathic Intellectual Disability Patients Ended with One Causative Anomaly; Duplication 9q34 Syndrome

Diagnostic Yield of Molecular Karyotyping of Idiopathic Intellectual Disability Patients Ended with One Causative Anomaly; Duplication 9q34 Syndrome

Detection of chromosomal rearrangements in the short arms of chromosomes 4 and 12 as an example of a whole-genome approach to noninvasive prenatal testing

Timely detection of fetal aneuploidy is an important aspect of clinical practice. At present, analytical techniques involving high-throughput sequencing are on the rise. Noninvasive prenatal testing (NIPT) ensures reliable results as early as week 9–11 into pregnancy. This article describes a clinical case of NIPT application and further verification of its results. Using next-generation sequencing, the microarray analysis of cell-free DNA in the amniotic fluid and the cytogenetic analysis of fetal chromosomes, a high risk of chromosomal rearrangements was detected in the short arms of chromosomes 4 and 12. This prediction was verified by molecular karyotyping conducted in both parents. The mother was found to be a balanced carrier of translocations between chromosomes 4 and 12. This case demonstrates the advantages of a whole-genome approach to NIPT over targeted-based.

Prenatal identification of partial 3q duplication syndrome

BackgroundThe 3q duplication syndrome is a result of duplication of a large fragment of the long arm of chromosome 3, mainly 3q21-qter, and in most cases it is diagnosed only after birth. The phenotypic consequences resulting from genetic imbalance are an important source of information for genetic counselling, especially in prenatal diagnostics. However, in most cases it is impossible to define them precisely because the final clinical presentation is a result of an overlap, usually due to different sizes of deletions and/or duplications not only chromosome 3 but also of translocation partner chromosome. In this article, we present a prenatal diagnosis of the 3q duplication syndrome in a foetus, arising from a balanced insertion ins (7,3)(q21.2;q12.3q29) carried by the mother.Case presentationThe article presents a case of a 29-year-old woman referred to the Genetic Outpatient Clinic for consultation in the 12th week of her fifth pregnancy with a diagnosis of generalised hydrops foetalis. The analysis of karyotype using GTG technique and FISH allowed diagnosis of a balanced aberration in the mother, and determined the type of chromosomal rearrangement, which allowed the identification of the origin of the additional genetic material in the foetus and the previous malformed child of the same couple. The use of molecular karyotyping techniques (FISH and aCGH) allowed a precise determination of the size of the imbalanced fragments in the affected siblings.ConclusionsThe aCGH technique is particularly valuable for the diagnostics of submicroscopic deletions and duplications, if no imbalanced chromosomal aberrations are detected by routine cytogenetic tests. It is also a valuable technique for identifying and fully characterizing genetic material of unknown origin, which can’t be identified using routine cytogenetic techniqes. However, it does not allow identification of balanced aberrations in carriers.

Microarray testing as an efficient tool to redefine hyperdiploid paediatric B-cell precursor acute lymphoblastic leukaemia patients

The aim of our study was to characterize genetic alterations in a cohort of paediatric patients with B-cell progenitors (BCP-ALL) and a hyperdiploid karyotype. In our study, we analysed 55 childhood hyperdiploid BCP-ALL patients using single nucleotide polymorphism (SNP) microarray testing. The group consisted mostly of patients with the modal number of chromosomes between 54 and 58 (34 cases). Within this group, Trisomy 4 and Trisomy 10 (30 cases) were the most frequent cases. Additionally, a total of 93 structural abnormalities mainly affecting chromosomes 1, 6, 9, 12, and 17 as well as 68 copy number alterations (CNAs) were identified. The microarray testing revealed a loss of ETV6, IKZF1, CDKN2A/CDKN2B, PAX5, and RB1. Moreover, chromosomal abnormalities resulting in the loss of heterozygosity (LOH) were also observed. Currently, patients with hyperdiploidy constitute a genetically heterogeneous group, and therefore, it is insufficient to rely only on banding cytogenetic analysis for the identification of hyperdiploid karyotype. Microarray testing has been proven an effective and satisfactory tool for the analysis of molecular karyotypes and to redefine the prognostic criteria in hyperdiploid patients.

BAP1‐deficient tumor/nevus with germline aberration: A potential pitfall in assessing melanocytic neoplasms with single nucleotide polymorphism array

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene, located on chromosome 3p21, encoding BAP1 nuclear protein, which is associated with a subset of melanocytic tumors with distinct cytologic features. Single nucleotide polymorphism array (SNP-array) is a molecular karyotyping technique that can detect copy number variations and loss of heterozygosity in various fresh and formalin-fixed paraffin-embedded tissues. Herein we present a 56-year-old female, who presented with a lesion on her left nose/cheek that was growing in size and changing in color. Histopathology was characteristic of a BAP1-deficient melanocytic neoplasm, with a biphasic population of cytologically bland conventional nevomelanocytes and a proliferation of large epithelioid melanocytes with abundant eosinophilic cytoplasm. Immunohistochemistry for BAP1 showed loss of nuclear labeling in the epithelioid melanocytes. SNP-array revealed a chromosome 21q22.1 monoaberration with no chromosome 3 abnormalities. The detection of this aberration prompted a discussion as to whether the lesion was best designated as a nevus or tumor. SNP-array on the patient's blood showed the same monoaberration of chromosome 21q22.1. This case emphasizes the importance of interpreting microarray results in the context of morphology, as germline aberrations can be a pitfall when assessing the genomic stability of a melanocytic proliferation by SNP array.

Induced pluripotent stem cell line heterozygous for p.R2447X mutation in filaggrin: KCLi002-A

We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.

Comprehensive analysis of isolated der(1;7)(q10;p10) in a large international homogenous cohort of patients with myelodysplastic syndromes.

The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the new comprehensive cytogenetic scoring system for MDS, chromosome 7 anomalies are no longer generally assigned to poor risk features but are thoroughly separated. However, der(1;7)(q10;p10), hereinafter der(1;7), is merged into the group labeled "any other single" and belongs to the intermediate risk group, just by definition due to lack of adequate clinical data. The aim of our international collaborative was to clarify the "real" prognostic impact of der(1;7) on a homogenous and well-documented data base. We performed detailed analysis of 63 MDS patients with isolated der(1;7) constituting the largest cohort hitherto reported. Furthermore, clinical data are compared with those of patients with isolated del(7q) and isolated monosomy 7. Median overall survival (OS) of patients with der(1;7) is 26 months (hazard ratio (HR) 0.91 for del(7q) vs der(1;7) and 2.53 for monosomy 7 vs der(1;7)). The der(1;7) is associated with profound thrombocytopenia most probably causing the reduced OS which is in striking contrast to the low risk for AML transformation (HR 3.89 for del(7q) vs der(1;7) and 5.88 for monosomy 7 vs der(1;7)). Molecular karyotyping indicates that der(1;7) is generated in a single step during mitosis and that a chromosomal imbalance rather than a single disrupted gene accounts for malignancy. Thus, the current cytogenetic scoring system assigning isolated der(1;7) to the intermediate risk group is now confirmed by a sufficient data set.

Exposure index of methyl isocyanate (MIC) gas disaster and a comprehensive spectrum of cytogenetic analysis after 30 years.

Severity of clinical expression and high mortality could not facilitate establishing exposure index/association following MIC disaster in Bhopal. Mortality-based exposure stratification was critiqued by the International Medical Commission on Bhopal (IMCB). IMCB stratified exposure considering distance as surrogate at 2km intervals after 10years. The first follow-up cytogenetic screening of the pre-screened survivors after 30years has demonstrated chromosome abnormalities (CA). Exposure stratification was attempted considering cytogenetic screening conducted during 1986-1988. Elevation of CA appeared proportional to exposure status and authenticated the initial mortality-based stratification. The one-on-one comparison of the previous and present cytogenetics has described the individual response to MIC exposure over 30years. Chi-square test has been carried out for checking the cytogenetic changes at the individual level statistically, which revealed that differences of chromosomal aberrations collected immediately post-disaster and 30years later are nonsignificant. The prominence of interindividual variation was noticed in general. The impact of overall exposure was higher in males. Constitutional abnormalities in 8.5% of the study population, including translocation, inversion, deletion, fragile sites, etc., necessitate screening of blood-linked members. The incidence of acrocentric association was prominent in the study population. Normal karyotype in children born to severely exposed parents with congenital anomalies indicates necessity of molecular karyotyping and/or screening of mutations. The study highlights follow-up of the health of the index cases at shorter (3-6months) intervals. This comprehensive spectrum of cytogenetic report highlights immediate post-disaster chromosomal aberrations, the changes that occurred over 30years in conjunction with other environmental factors at the individual level, constitutive genomic aberrations, polymorphic variations, and chromosomal patterns in congenitally malformed children of the survivors, which collectively indicate the possibility of acquisition/persistence of stable aberrations in MIC-exposed lymphocytes through interaction with environmental/biological confounders.

The frequency of CNVs in a cohort population of consecutive fetuses with congenital anomalies after the termination of pregnancy.

BackgroundThe implementation of molecular karyotyping has resulted in an improved diagnostic yield in the genetic diagnostics of congenital anomalies, detected prenatally or after the termination of pregnancy. However, the systematic epidemiologic ascertainment of copy number variations in the etiology of congenital anomalies has not yet been sufficiently explored.MethodsConsecutive fetuses, altogether 204, with major single or multiple congenital anomalies were ascertained by using the SLOCAT registry for the period from 2011 to 2015. After excluding aneuploidies by using conventional karyotyping or Quantitative Fluorescence‐Polymerase Chain Reaction, array comparative genomic hybridization was performed for the detection of copy number variations.ResultsWe identified pathogenic or likely pathogenic copy number variations in 14 fetuses (6.8%); 2.9% in fetuses with isolated, and 3.9% in fetuses with multiple congenital anomalies. Additionally, aneuploidies and major structural chromosomal abnormalities were detected in 40.2%.ConclusionOur systematic approach of ascertaining congenital anomalies resulted in explaining the etiology of congenital anomalies in 47% of fetuses after the termination of pregnancy. By using array comparative genomic hybridization, we found that copy number variations represent an important part in the etiology of multiple, as well as isolated congenital anomalies, which indicates the importance of analyzing copy number variations in the diagnostic approach of fetuses with congenital anomalies after the termination of pregnancy.

Interstitial 14q31.3-q32.13 deletion

Aim: With the exception of ring chromosome 14 or translocations, interstitial deletions of the long arm of chromosome 14 are very rare. All patients with these deletions share common phenotypic characteristics, primarily mild dysmorphia and developmental delay. Molecular karyotyping (array CGH) enabled the precise breakpoint determination and improved the analysis of genotype-phenotype correlations. Case presentation: In a 7-year-old girl, array CGH was performed due to developmental delay. The array CGH study showed 8.3Mb de novo interstitial deletion of the 14q31.3–q32.13 region. Conclusions: Comparison of our patient´s phenotype with previously reported chromosome 14q interstitial deletion cases confirmed the presence of common clinical features and highlights the utility of array CGH as a diagnostic tool in clarifying the developmental delay etiology.

Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

Chromosomal microarray comparative genome hybridization (arrayCGH) in prenatal settings. Proposal for Hungarian application in clinical practice

Invasive prenatal testing and conventional G-banding chromosome analysis have been considered to be the gold standard of fetal cytogenetic diagnosis. Standard karyotyping is, however, constrained by the limits of the resolution of using a microscope. The advantage of molecular karyotyping, array based methods is the evaluation of sub-microscopic copy number changes across the whole genome in a single analysis. The application of array comparative genome hybridization has greatly increased the detection of pathogenic chromosomal abnormalities in prenatal settings. Based on available data in the international literature of the last decade, the clinical utility of arrayCGH is the recognition of some 1-2% and 5-7% additional genetical information compared to metaphase karyotype alone in fetuses without ultrasound anomaly and in fetuses with ultrasonographically detected malformations, respectively. Thus arrayCGH improves the prenatal diagnosis of genetic abnormalities mainly in fetuses with structural sonographic findings. In the present paper we review the literature of chromosomal microarray and make a proposal for the application of the method in Hungarian prenatal genetical practice. Orv Hetil. 2019; 160(13): 484-493.

Unbalanced X;9 translocation in an infertile male with de novo duplication Xp22.31p22.33.

Male carriers of an X-autosome translocation are generally infertile, regardless of the position of the breakpoint on the X chromosome while the pathogenicity of Xp22.3 subtelomeric duplications is under debate. To shed light into this controversy, we present a rare case, of an azoospermic male with no other significant clinical findings, in whom classical cytogenetics revealed additional unbalanced chromosomal material, at the telomere of the long arm of one homolog of chromosome 9. In peripheral blood specimens of the index case and his parents, we performed GBanding, Inverted-DAPI Banding, AgNOR staining, Telomere specific Fluorescence in Situ Hybridization (FISH), Molecular karyotyping by Multi-color FISH, whole genome SNP microarrays, sub-telomeric MLPA, and transcription analysis of the expression of KAL1 gene by RT-PCR. Multi-color FISH revealed an unbalanced translocation involving the short arm of chromosome X. SNP microarray analysis combined to classical cytogenetics and MLPA demonstrated a de novo 8.796Mb duplication of Xp22.31-p22.33. Compared to three control specimens, the patient presented significantly elevated expression levels of KAL1 mRNA in peripheral blood, suggesting transcriptional functionality of the duplicated segment. The duplicated segment contains the pseudo-autosomal region PAR1 and more than 30 genes including SHOX, ARSE, STS, KAL1, and FAM9A and is not listed as polymorphic. Our data advocate that duplications of the Xp22.3 region may not be associated with a clinical consequence.

Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola

BackgroundThe apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the parthenogenesis component of apomixis in Pennisetum squamulatum, was diagnostic for reproductive mode in the closely related species B. ruziziensis, B. brizantha, and B. decumbens. In this study, we used a mapping population of the distantly related commercial species B. humidicola to map the ASGR and test for conservation of ASGR-BBML sequences across Brachiaria species.ResultsDense genetic maps were constructed for the maternal and paternal genomes of a hexaploid (2n = 6x = 36) B. humidicola F1 mapping population (n = 102) using genotyping-by-sequencing, simple sequence repeat, amplified fragment length polymorphism, and transcriptome derived single nucleotide polymorphism markers. Comparative genomics with Setaria italica provided confirmation for x = 6 as the base chromosome number of B. humidicola. High resolution molecular karyotyping indicated that the six homologous chromosomes of the sexual female parent paired at random, whereas preferential pairing of subgenomes was observed in the apomictic male parent. Furthermore, evidence for compensated aneuploidy was found in the apomictic parent, with only five homologous linkage groups identified for chromosome 5 and seven homologous linkage groups of chromosome 6. The ASGR mapped to B. humidicola chromosome 1, a region syntenic with chromosomes 1 and 7 of S. italica. The ASGR-BBML specific PCR product cosegregated with the ASGR in the F1 mapping population, despite its location on a different carrier chromosome than B. decumbens.ConclusionsThe first dense molecular maps of B. humidicola provide strong support for cytogenetic evidence indicating a base chromosome number of six in this species. Furthermore, these results show conservation of the ASGR across the Paniceae in different chromosomal backgrounds and support postulation of the ASGR-BBML as candidate genes for the parthenogenesis component of apomixis.

Familial Xp11.22 microdeletion including SHROOM4 and CLCN5 is associated with intellectual disability, short stature, microcephaly and Dent disease: a case report

BackgroundTwo interstitial microdeletions Xp11.22 including the CLCN5 and SHROOM4 genes were recently reported in a male individual affected with Dent disease, short stature, psychomotor delay and minor facial anomalies. Dent disease, characterized by a specific renal phenotype, is caused by truncating mutations of CLCN5 in the majority of affected cases.Case presentationHere, we present clinical and molecular findings in a male patient with clinical signs of Dent disease, developmental delay, short stature, microcephaly, and facial dysmorphism. Using molecular karyotyping we identified a hemizygous interstitial microdeletion Xp11.23p.11.22 of about 700 kb, which was inherited from his asymptomatic mother. Among the six deleted genes is CLCN5, which explains the renal phenotype in our patient. SHROOM4, which is partially deleted in this patient, is involved in neuronal development and was shown to be associated with X-linked intellectual disability. This is a candidate gene, the loss of which is thought to be associated with his further clinical manifestations.To rule out mutations in other genes related to intellectual disability, whole exome sequencing was performed. No other pathogenic variants that could explain the phenotypic features, were found.ConclusionWe compared the clinical findings of the patient presented here with the reported case with an Xp11.22 microdeletion including CLCN5 and SHROOM4 and re-defined the phenotypic spectrum associated with this microdeletion.