Variants in Candidate Genes for Phenotype Heterogeneity in Patients with the 22q11.2 Deletion Syndrome.

22q11.2 deletion syndrome (22q11.2DS) is a microdeletion syndrome with a broad and heterogeneous phenotype, even though most of the deletions present similar sizes, involving ∼3 Mb of DNA. In a relatively large population of a Brazilian 22q11.2DS cohort (60 patients), we investigated genetic variants that could act as genetic modifiers and contribute to the phenotypic heterogeneity, using a targeted NGS (Next Generation Sequencing) with a specific Ion AmpliSeq panel to sequence nine candidate genes (CRKL, MAPK1, HIRA, TANGO2, PI4KA, HDAC1, ZDHHC8, ZFPM2, and JAM3), mapped in and outside the 22q11.2 hemizygous deleted region. In silico prediction was performed, and the whole-genome sequencing annotation analysis package (WGSA) was used to predict the possible pathogenic effect of single nucleotide variants (SNVs). For the in silico prediction of the indels, we used the genomic variants filtered by a deep learning model in NGS (GARFIELD-NGS). We identified six variants, 4 SNVs and 2 indels, in MAPK1, JAM3, and ZFPM2 genes with possibly synergistic deleterious effects in the context of the 22q11.2 deletion. Our results provide the opportunity for the discovery of the co-occurrence of genetic variants with 22q11.2 deletions, which may influence the patients´ phenotype.

Variants in KMT2A in Three Individuals with Previous Suspicion of 22q11.2 Deletion Syndrome.

The condition known as 22q11.2 deletion syndrome (MIM #188400) is a rare disease with a highly variable clinical presentation including more than 180 features; specific guidelines for screening individuals have been used to support clinical suspicion before confirmatory tests by Brazil's Craniofacial Project. Of the 2568 patients listed in the Brazilian Database on Craniofacial Anomalies, 43 individuals negative for the 22q11.2 deletion syndrome were further investigated through whole-exome sequencing. Three patients (6.7%) presented with heterozygous pathogenic variants in the KMT2A gene, including a novel variant (c.6158+1del) and two that had been previously reported (c.173dup and c.3241C>T); reverse phenotyping concluded that all three patients presented features of Wiedemann-Steiner syndrome, such as neurodevelopmental disorders and dysmorphic facial features (n = 3), hyperactivity and anxiety (n = 2), thick eyebrows and lower-limb hypertrichosis (n = 2), congenital heart disease (n = 1), short stature (n = 1), and velopharyngeal insufficiency (n = 2). Overlapping features between 22q11.2 deletion syndrome and Wiedemann-Steiner syndrome comprised neuropsychiatric disorders and dysmorphic characteristics involving the eyes and nose region; velopharyngeal insufficiency was seen in two patients and is an unreported finding in WDSTS. Therefore, we suggest that both conditions should be included in each other's differential diagnoses.

Premature ovarian insufficiency is associated with global alterations in the regulatory landscape and gene expression in balanced X-autosome translocations.

BACKGROUND: Patients with balanced X-autosome translocations and premature ovarian insufficiency (POI) constitute an interesting paradigm to study the effect of chromosome repositioning. Their breakpoints are clustered within cytobands Xq13-Xq21, 80% of them in Xq21, and usually, no gene disruption can be associated with POI phenotype. As deletions within Xq21 do not cause POI, and since different breakpoints and translocations with different autosomes lead to this same gonadal phenotype, a "position effect" is hypothesized as a possible mechanism underlying POI pathogenesis. OBJECTIVE AND METHODS: To study the effect of the balanced X-autosome translocations that result in POI, we fine-mapped the breakpoints in six patients with POI and balanced X-autosome translocations and addressed gene expression and chromatin accessibility changes in four of them. RESULTS: We observed differential expression in 85 coding genes, associated with protein regulation, multicellular regulation, integrin signaling, and immune response pathways, and 120 differential peaks for the three interrogated histone marks, most of which were mapped in high-activity chromatin state regions. The integrative analysis between transcriptome and chromatin data pointed to 12 peaks mapped less than 2 Mb from 11 differentially expressed genes in genomic regions not related to the patients' chromosomal rearrangement, suggesting that translocations have broad effects on the chromatin structure. CONCLUSION: Since a wide impact on gene regulation was observed in patients, our results observed in this study support the hypothesis of position effect as a pathogenic mechanism for premature ovarian insufficiency associated with X-autosome translocations. This work emphasizes the relevance of chromatin changes in structural variation, since it advances our knowledge of the impact of perturbations in the regulatory landscape within interphase nuclei, resulting in the position effect pathogenicity.

The effect of brain-derived neurotrophic factor Val66Met polymorphism on adolescent activity and rest rhythms, circadian preferences and attentional performance.

BACKGROUND: Variations in circadian regulating mechanisms generate different individual preferences in respect of sleep and activity timing, which are known as chronotypes. In this sense, specifically during adolescence, there is a greater tendency for an eveningness chronotype. One factor that has been shown to have an impact on circadian rhythm patterns, as well as on some aspects of cognitive function, is the relatively common Val66Met (rs6265) polymorphism in the human brain-derived neurotrophic factor gene. OBJECTIVE: This study aimed to evaluate the effect of the BDNF Val66Met polymorphism on the performance of adolescents in attentional tests, circadian preferences and activity-rest rhythm. METHODS: 85 healthy high school students completed the Morningness-Eveningness Questionnaire to assess their circadian preferences; were evaluated using the Psychological Battery for Attention Assessment; and were categorized as carriers and non-carriers of the rs6265 polymorphism using the TaqMan rt-PCR technique. A subsample of 42 students had their activity/rest rhythm recorded by actigraphy for nine days from which sleep parameters were estimated. RESULTS: Circadian preference did not affect attentional performance (p > 0.1), but the time that the students attended school had an impact on all types of attention with morning shift students scoring higher, regardless of chronotype (p < 0.05). The presence of the BDNF Val66Met polymorphism was associated only with alternate attention performance (p < 0.05). Regarding actigraphy evaluation, the carriers of the polymorphism demonstrated significantly higher total time in bed, total sleep time, social jetlag, and earlier sleep onset. CONCLUSIONS: The results indicate some degree of adaptation in the students' attentional performance, according to their school schedules. The presence of BDNF polymorphism demonstrated a counterintuitive impact on attentional performance, comparing to previous findings. The findings reinforce the effect of genetic traits on sleep-wake rhythm parameters, when objectively evaluated.

Fold-back mechanism originating inv-dup-del rearrangements in chromosomes 13 and 15.

Intrachromosomal rearrangements involve a single chromosome and can be formed by several proposed mechanisms. We reported two patients with intrachromosomal duplications and deletions, whose rearrangements and breakpoints were characterized through karyotyping, chromosomal microarray, fluorescence in situ hybridization, whole-genome sequencing, and Sanger sequencing. Inverted duplications associated with terminal deletions, known as inv-dup-del rearrangements, were found in 13q and 15q in these patients. The presence of microhomology at the junction points led to the proposal of the Fold-back mechanism for their formation. The use of different high-resolution techniques allowed for a better characterization of the rearrangements, with Sanger sequencing of the junction points being essential to infer the mechanisms of formation as it revealed microhomologies that were missed by the previous techniques. A karyotype-phenotype correlation was also performed for the characterized rearrangements.

The impact of 22q11.2 copy-number variants on human traits in the general population.

While extensively studied in clinical cohorts, the phenotypic consequences of 22q11.2 copy-number variants (CNVs) in the general population remain understudied. To address this gap, we performed a phenome-wide association scan in 405,324 unrelated UK Biobank (UKBB) participants by using CNV calls from genotyping array. We mapped 236 Human Phenotype Ontology terms linked to any of the 90 genes encompassed by the region to 170 UKBB traits and assessed the association between these traits and the copy-number state of 504 genotyping array probes in the region. We found significant associations for eight continuous and nine binary traits associated under different models (duplication-only, deletion-only, U-shape, and mirror models). The causal effect of the expression level of 22q11.2 genes on associated traits was assessed through transcriptome-wide Mendelian randomization (TWMR), revealing that increased expression of ARVCF increased BMI. Similarly, increased DGCR6 expression causally reduced mean platelet volume, in line with the corresponding CNV effect. Furthermore, cross-trait multivariable Mendelian randomization (MVMR) suggested a predominant role of genuine (horizontal) pleiotropy in the CNV region. Our findings show that within the general population, 22q11.2 CNVs are associated with traits previously linked to genes in the region, and duplications and deletions act upon traits in different fashions. We also showed that gain or loss of distinct segments within 22q11.2 may impact a trait under different association models. Our results have provided new insights to help further the understanding of the complex 22q11.2 region.

X-Chromosome Dependent Differences in the Neuronal Molecular Signatures and Their Implications in Sleep Patterns.

Biological factors and mechanisms that drive sex differences observed in sleep disturbances are understudied and poorly understood. The extent to which sex chromosome constitution impacts on sex differences in circadian patterns is still a knowledge void in the sleep medicine field. Here we focus on the neurological consequences of X-chromosome functional imbalances between males and females and how this molecular inequality might affect sex divergencies on sleep. In light of the X-chromosome inactivation mechanism in females and its implications in gene regulation, we describe sleep-related neuronal circuits and brain regions impacted by sex-biased modulations of the transcriptome and the epigenome. Benefited from recent large-scale genetic studies on the interplay between X-chromosome and brain function, we list clinically relevant genes that might play a role in sex differences in neuronal pathways. Those molecular signatures are put into the context of sleep and sleep-associated neurological phenotypes, aiming to identify biological mechanisms that link X-chromosome gene regulation to sex-biased human traits. These findings are a significant step forward in understanding how X-linked genes manifest in sleep-associated transcriptional networks and point to future research opportunities to address female-specific clinical manifestations and therapeutic responses.

Allan-Herndon-Dudley syndrome in a female patient and related mechanisms.

Allan-Herndon-Dudley syndrome (AHDS) is characterized by neuropsychomotor developmental delay/intellectual disability, neurological impairment with a movement disorder, and an abnormal thyroid hormone profile. This disease is an X-linked disorder that mainly affects men. We described a female patient with a de novo variant in the SLC16A2 gene, a milder AHDS phenotype, and a skewed X chromosome inactivation profile. We discuss the mechanisms associated with the expression of the phenotypic characteristics in female patients, including SLC16A2 gene variants and cytogenomic alterations, as well as preferential inactivation of the normal X chromosome.

Mechanisms of structural chromosomal rearrangement formation.

Structural chromosomal rearrangements result from different mechanisms of formation, usually related to certain genomic architectural features that may lead to genetic instability. Most of these rearrangements arise from recombination, repair, or replication mechanisms that occur after a double-strand break or the stalling/breakage of a replication fork. Here, we review the mechanisms of formation of structural rearrangements, highlighting their main features and differences. The most important mechanisms of constitutional chromosomal alterations are discussed, including Non-Allelic Homologous Recombination (NAHR), Non-Homologous End-Joining (NHEJ), Fork Stalling and Template Switching (FoSTeS), and Microhomology-Mediated Break-Induced Replication (MMBIR). Their involvement in chromoanagenesis and in the formation of complex chromosomal rearrangements, inverted duplications associated with terminal deletions, and ring chromosomes is also outlined. We reinforce the importance of high-resolution analysis to determine the DNA sequence at, and near, their breakpoints in order to infer the mechanisms of formation of structural rearrangements and to reveal how cells respond to DNA damage and repair broken ends.

Minimal Critical Region and Genes for a Typical Presentation of Langer-Giedion Syndrome.

Langer-Giedion syndrome (LGS) is caused by a contiguous deletion at 8q23q24, characterized by exostoses, facial, ectodermal, and skeletal anomalies, and, occasionally, intellectual disability. LGS patients have been diagnosed clinically or by routine cytogenetic techniques, hampering the definition of an accurate genotype-phenotype correlation for the syndrome. We report two unrelated patients with 8q23q24 deletions, characterized by cytogenomic techniques, with one of them, to our knowledge, carrying the smallest deletion reported in classic LGS cases. We assessed the pathogenicity of the deletion of genes within the 8q23q24 region and reviewed other molecularly confirmed cases from the literature. Our findings suggest a 3.2-Mb critical region for a typical presentation of the syndrome, emphasizing the contribution of the TRPS1, RAD21, and EXT1 genes' haploinsufficiency, and facial dysmorphisms as well as bone anomalies as the most frequent features among patients with LGS. We also suggest a possible role for the CSMD3 gene, whose deletion seems to contribute to central nervous system anomalies. Since studies performing such correlation for LGS patients are limited, our data contribute to improving the ge-notype-phenotype characterization for LGS patients.

Multicentric Carpotarsal Osteolysis Syndrome in a Mother and Daughter with a MAFB Missense Variant and Natural History of the Disease.

Multicentric carpotarsal osteolysis syndrome (MCTO; MIM #166300) is a rare skeletal disorder characterized by osteolysis affecting particularly the carpal, metacarpal, and tarsal bones, although other bones might be involved. MCTO is an autosomal dominant disease caused by heterozygous variants in the MAFB gene, frequently misdiagnosed as juvenile rheumatoid arthritis due to similar clinical manifestations. This study reports the first Brazilian family diagnosed with MCTO with progressive osteolysis of the carpal and tarsal bones, presenting a c.161C>T (p.Ser54Leu) heterozygous variant in the MAFB gene, describing the clinical, radiological, and molecular findings, compared with literature data, and discussing the different clinical and molecular diagnosis, as well as the natural history of the disease. Since MCTO is a disorder with progressive symptoms, an early diagnosis is important to avoid unnecessary investigations and treatments and to provide the proper follow-up.

CEDNIK syndrome in a Brazilian patient with compound heterozygous pathogenic variants.

CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis, and Keratoderma) syndrome is a neuro ichthyotic syndrome characterized by a clinical constellation of features including severe developmental delay, microcephaly, and facial dysmorphism. Here, we report the clinical and molecular characterization of a patient with CEDNIK syndrome harboring two compound heterozygous variants in the SNAP29 gene. The patient presents a combination of a loss-of-function SNAP29 mutation and a ∼370 kb 22q11.2 deletion, each of these genetic variants inherited from one of the parents. This report provides detailed data of a patient with unprecedented genetic events leading to the CEDNIK phenotype and may contribute to the elucidation of this rare condition.

Intelligence Quotient Variability in Klinefelter Syndrome Is Associated With GTPBP6 Expression Under Regulation of X-Chromosome Inactivation Pattern.

Klinefelter syndrome (KS) displays a broad dysmorphological, endocrinological, and neuropsychological clinical spectrum. We hypothesized that the neurocognitive dysfunction present in KS relies on an imbalance in X-chromosome gene expression. Thus, the X-chromosome inactivation (XCI) pattern and neurocognitive X-linked gene expression were tested and correlated with intelligence quotient (IQ) scores. We evaluated 11 KS patients by (a) IQ assessment, (b) analyzing the XCI patterns using both HUMARA and ZDHHC15 gene assays, and (c) blood RT-qPCR to investigate seven X-linked genes related to neurocognitive development (GTPBP6, EIF2S3, ITM2A, HUWE1, KDM5C, GDI1, and VAMP7) and XIST in comparison with 14 (male and female) controls. Considering IQ 80 as the standard minimum reference, we verified that the variability in IQ scores in KS patients seemed to be associated with the XCI pattern. Seven individuals in the KS group presented a random X-inactivation (RXI) and lower average IQ than the four individuals who presented a skewed X-inactivation (SXI) pattern. The evaluation of gene expression showed higher GTPBP6 expression in KS patients with RXI than in controls (p = 0.0059). Interestingly, the expression of GTPBP6 in KS patients with SXI did not differ from that observed in controls. Therefore, our data suggest for the first time that GTPBP6 expression is negatively associated with full-scale IQ under the regulation of the type of XCI pattern. The SXI pattern may regulate GTPBP6 expression, thereby dampening the impairment in cognitive performance and playing a role in intelligence variability in individuals with KS, which warrants further mechanistic investigations.

Spread of X-chromosome inactivation into autosomal regions in patients with unbalanced X-autosome translocations and its phenotypic effects.

Patients with unbalanced X-autosome translocations are rare and usually present a skewed X-chromosome inactivation (XCI) pattern, with the derivative chromosome being preferentially inactivated, and with a possible spread of XCI into the autosomal regions attached to it, which can inactivate autosomal genes and affect the patients' phenotype. We describe three patients carrying different unbalanced X-autosome translocations, confirmed by G-banding karyotype and array techniques. We analyzed their XCI pattern and inactivation spread into autosomal regions, through HUMARA, ZDHHC15 gene assay and the novel 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and identified an extremely skewed XCI pattern toward the derivative chromosomes for all the patients, and a variable pattern of late-replication on the autosomal regions of the derivative chromosomes. All patients showed phenotypical overlap with patients presenting deletions of the autosomal late-replicating regions, suggesting that the inactivation of autosomal segments may be responsible for their phenotype. Our data highlight the importance of the XCI spread into autosomal regions for establishing the clinical picture in patients carrying unbalanced X-autosome translocations, and the incorporation of EdU as a novel and precise tool to evaluate the inactivation status in such patients.

Novel MYT1 variants expose the complexity of oculo-auriculo-vertebral spectrum genetic mechanisms.

Oculo-auriculo-vertebral spectrum (OAVS) is a developmental disorder characterized by anomalies mainly involving the structures derived from the first and second pharyngeal arches. The spectrum presents with heterogeneous clinical features and complex etiology with genetic factors not yet completely understood. To date, MYT1 is the most important gene unambiguously associated with the spectrum and with functional data confirmation. In this work, we aimed to identify new single nucleotide variants (SNVs) affecting MYT1 in a cohort of 73 Brazilian patients diagnosed with OAVS. In addition, we investigated copy number variations (CNVs) encompassing this gene or its cis-regulatory elements and compared the frequency of these events in patients versus a cohort of 455 Brazilian control individuals. A new SNV, predicted as likely deleterious, was identified in five unrelated patients with OAVS. All five patients presented hearing impairment and orbital asymmetry suggesting an association with the variant. CNVs near MYT1, located in its neighboring topologically associating domain (TAD), were found to be enriched in patients when compared to controls, indicating a possible involvement of this region with OAVS pathogenicity. Our findings highlight the genetic complexity of the spectrum that seems to involve more than one variant type and inheritance patterns.

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco's Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (±4.5) days, with an average cellular yield of 12.3 (± 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (±24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

Copy number variation (CNV) identification, interpretation, and database from Brazilian patients.

Copy number variations (CNVs) constitute an important class of variation in the human genome and the interpretation of their pathogenicity considering different frequencies across populations is still a challenge for geneticists. Since the CNV databases are predominantly composed of European and non-admixed individuals, and Brazilian genetic constitution is admixed and ethnically diverse, diagnostic screenings on Brazilian variants are greatly difficulted by the lack of populational references. We analyzed a clinical sample of 268 Brazilian individuals, including patients with neurodevelopment disorders and/or congenital malformations. The pathogenicity of CNVs was classified according to their gene content and overlap with known benign and pathogenic variants. A total of 1,504 autosomal CNVs (1,207 gains and 297 losses) were classified as benign (92.9%), likely benign (1.6%), VUS (2.6%), likely pathogenic (0.2%) and pathogenic (2.7%). Some of the CNVs were recurrent and with frequency increased in our sample, when compared to populational open resources of structural variants: 14q32.33, 22q11.22, 1q21.1, and 1p36.32 gains. Thus, these highly recurrent CNVs classified as likely benign or VUS were considered non-pathogenic in our Brazilian sample. This study shows the relevance of introducing CNV data from diverse cohorts to improve on the interpretation of clinical impact of genomic variations.

Lymphoproliferative disorder with polyautoimmunity and hypogammaglobulinemia: An unusual presentation of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) has a heterogeneous presentation that includes multiple congenital anomalies and immunodeficiency, one of the most striking features. Usually, it is characterized by T cell lymphopenia, B cell dysfunction and autoimmunity. Here, we describe an unusual case of 22q11.2DS in a patient with lymphoproliferative disorder, polyautoimmunity and hypogammaglobulinemia.

Genetics of premature ovarian insufficiency and the association with X-autosome translocations.

Premature ovarian insufficiency (POI) is the cessation of menstruation before the age of 40 and can result from different etiologies, including genetic, autoimmune, and iatrogenic. Of the genetic causes, single-gene mutations and cytogenetic alterations, such as X-chromosome aneuploidies and chromosome rearrangements, can be associated with POI. In this review, we summarize the genetic factors linked to POI and list the main candidate genes. We discuss the association of these genes with the ovarian development, the functional consequences of different mutational mechanisms and biological processes that are frequently disrupted during POI pathogenesis. Additionally, we focus on the high prevalence of X-autosome translocations involving the critical regions in Xq, known as POI1 and POI2, and ddiscuss in depth the main hypotheses proposed to explain this association. Although the incorrect pairing of chromosomes during meiosis could lead to oocyte apoptosis, the reason for the prevalence of X-chromosome breakpoints at specific regions remains unclear. In most cases, studies on genes disrupted by balanced structural rearrangements cannot explain the ovarian failure. Thus, the position effect has emerged as a putative explanation for genetic mechanisms as translocations possibly result in changes in overall chromatin topology due to chromosome repositioning. Given the tremendous impact of POI on women's quality of life, we highlight the value of investigations in to the interplay between ovarian function and gene regulation to deepen our understanding of the molecular mechanisms related to this disease, with the ultimate goal of improving patients' care and assistance.