Investigating the correlation between genotype and phenotype in Prader-Willi syndrome: a study of 45 cases from Brazil.

BACKGROUND: Prader-Willi syndrome (PWS) is a genetic disorder characterized by abnormalities in the 15q11-q13 region. Understanding the correlation between genotype and phenotype in PWS is crucial for improved genetic counseling and prognosis. In this study, we aimed to investigate the correlation between genotype and phenotype in 45 PWS patients who previously underwent methylation-sensitive high-resolution melting (MS-HRM) for diagnosis. RESULTS: We employed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and Sanger sequencing, along with collecting phenotypic data from the patients for comparison. Among the 45 patients, 29 (64%) exhibited a deletion of 15q11-q13, while the remaining 16 (36%) had uniparental disomy. No statistically significant differences were found in the main signs and symptoms of PWS. However, three clinical features showed significant differences between the groups. Deletion patients had a higher prevalence of myopia than those with uniparental disomy, as well as obstructive sleep apnea and an unusual skill with puzzles. CONCLUSIONS: The diagnostic tests (MS-HRM, MS-MLPA, and Sanger sequencing) yielded positive results, supporting their applicability in PWS diagnosis. The study's findings indicate a general similarity in the genotype-phenotype correlation across genetic subtypes of PWS.

A cohort study of neurodevelopmental disorders and/or congenital anomalies using high resolution chromosomal microarrays in southern Brazil highlighting the significance of ASD.

Chromosomal microarray (CMA) is the reference in evaluation of copy number variations (CNVs) in individuals with neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and/or autism spectrum disorder (ASD), which affect around 3-4% of the world's population. Modern platforms for CMA, also include probes for single nucleotide polymorphisms (SNPs) that detect homozygous regions in the genome, such as long contiguous stretches of homozygosity (LCSH). These regions result from complete or segmental chromosomal homozygosis and may be indicative of uniparental disomy (UPD), inbreeding, population characteristics, as well as replicative DNA repair events. In this retrospective study, we analyzed CMA reading files requested by geneticists and neurologists for diagnostic purposes along with available clinical data. Our objectives were interpreting CNVs and assess the frequencies and implications of LCSH detected by Affymetrix CytoScan HD (41%) or 750K (59%) platforms in 1012 patients from the south of Brazil. The patients were mainly children with NDDs and/or congenital anomalies (CAs). A total of 206 CNVs, comprising 132 deletions and 74 duplications, interpreted as pathogenic, were found in 17% of the patients in the cohort and across all chromosomes. Additionally, 12% presented rare variants of uncertain clinical significance, including LPCNVs, as the only clinically relevant CNV. Within the realm of NDDs, ASD carries a particular importance, owing to its escalating prevalence and its growing repercussions for individuals, families, and communities. ASD was one clinical phenotype, if not the main reason for referral to testing, for about one-third of the cohort, and these patients were further analyzed as a sub-cohort. Considering only the patients with ASD, the diagnostic rate was 10%, within the range reported in the literature (8-21%). It was higher (16%) when associated with dysmorphic features and lower (7%) for "isolated" ASD (without ID and without dysmorphic features). In 953 CMAs of the whole cohort, LCSH (≥ 3 Mbp) were analyzed not only for their potential pathogenic significance but were also explored to identify common LCSH in the South Brazilians population. CMA revealed at least one LCSH in 91% of the patients. For about 11.5% of patients, the LCSH suggested consanguinity from the first to the fifth degree, with a greater probability of clinical impact, and in 2.8%, they revealed a putative UPD. LCSH found at a frequency of 5% or more were considered common LCSH in the general population, allowing us to delineate 10 regions as potentially representing ancestral haplotypes of neglectable clinical significance. The main referrals for CMA were developmental delay (56%), ID (33%), ASD (33%) and syndromic features (56%). Some phenotypes in this population may be predictive of a higher probability of indicating a carrier of a pathogenic CNV. Here, we present the largest report of CMA data in a cohort with NDDs and/or CAs from the South of Brazil. We characterize the rare CNVs found along with the main phenotypes presented by each patient and show the importance and usefulness of LCSH interpretation in CMA results that incorporate SNPs, as well as we illustrate the value of CMA to investigate CNV in ASD.

[Prader-Willi and Angelman syndromes: case series diagnosed by MS-MLPA assay]. / Síndromes Prader-Willi y Angelman: serie de casos diagnosticados mediante la técnica de MS-MLPA.

BACKGROUND: Prader Willi syndrome (PWS) and Angelman syndrome (AS) are neurodevelopmental disorders caused by deletions or methylation defects, making a loss of expression of imprinted genes located in the 15q11-q13 region, and these can be assessed by different cytogenomic and molecular techniques. We report a case series of patients with PWS and AS evaluated through the MS-MLPA assay. CLINICAL CASES: We studied four patients with a clinical diagnosis of PWS and another with AS, evaluated as far as possible with karyotype and FISH, and with MS-MLPA assay for the 15q11-q13 region in all cases. In patients with PWS, neonatal hypotonia was the main reason for consultation and in three of them we identified a deletion of 15q11-q13 by MS-MLPA, also confirmed by FISH; and in the other one, an abnormal methylation pattern consistent with a maternal uniparental disomy. The patient with AS presented with a typical picture which led to the identification of a deletion in 15q11-q13 by MS-MLPA, also confirmed by FISH. CONCLUSIONS: The use of the MS-MLPA assay for the 15q11-q13 region was very useful for the diagnosis and identification of the genomic and epigenetic defects involved in either PWS and AS.

INTRODUCCIÓN: el síndrome Prader-Willi (SPW) y el síndrome de Angelman (SA) son trastornos del neurodesarrollo producidos por deleciones o defectos de metilación que producen pérdida de expresión en los genes improntados de la región 15q11 q13, mismos que pueden ser evaluados por diferentes técnicas citogenómicas y moleculares. Presentamos una serie de pacientes con SPW y SA en los que se identificó el tipo de defecto de la región 15q11-q13 mediante la técnica de MS-MLPA. CASOS CLÍNICOS: estudiamos cuatro pacientes con diagnóstico clínico de SPW y uno con SA, evaluados en lo posible con cariotipo, FISH y todos con ensayo MS-MLPA para la región 15q11-q13. En los pacientes con SPW, la hipotonía neonatal fue el motivo principal de consulta. En tres de ellos se identificó deleción de 15q11-q13 por MS-MLPA, confirmada por FISH, y en uno el patrón de metilación anormal fue compatible con una disomía uniparental materna. El paciente con SA presentó un cuadró típico y también se identificó una deleción en 15q11-q13 por MS-MLPA, confirmada por FISH. CONCLUSIONES: confirmamos que el uso de la técnica de MS-MLPA para la región 15q11 q13 mostró ser de gran utilidad para identificar los mecanismos genómicos y epigenéticos implicados en el SPW y el SA.

Analysis of an NGS retinopathy panel detects chromosome 1 uniparental isodisomy in a patient with RPE65-related leber congenital amaurosis.

Purpose: This study aims to demonstrate the possibility of detecting segmental uniparental isodisomy (iUPD) using a next-generation sequencing gene panel by reporting a Leber congenital amaurosis (LCA) case caused by a homozygous pathogenic variant in RPE65 (c.1022 T > C:p.Leu341Ser) inherited exclusively from the proband's mother.Methods: Samples from the trio (proband, mother, and father) were sequenced with a next-generation sequencing (NGS) retinopathy gene panel (224 genes) and the VCF file containing all variants was used in order to determine single nucleotide variant (SNV) counts from each sample across all chromosomes.Results: Trio analysis showed that of 81 Chr1 inherited variants 41 were exclusively maternal, including 21 homozygous. The other 40 variants were common to both parents. On remaining autosomal chromosomes (Chr2-22) 645 inherited variants were found, 147 of them were exclusively maternal and 132 exclusively paternal. Based on these NGS data, it was possible to note that the proband's chromosomes 1 are more similar to his mother's chromosome 1 than his father's, suggesting the pathogenic homozygous variant found in this patient was inherited exclusively from the mother due to uniparental maternal isodisomy.Conclusions: This study presents a secondary analysis pipeline to identify responsible variants for a phenotype and the correct inheritance pattern, which is a critical step to the proper and accurate genetic counseling of all family members. In addition, this approach could be used to determine iUPD in different Mendelian disorders if the sequencing panel identifies variants spread throughout the genome.

Evolution over Time of Leg Length Discrepancy in Patients with Syndromic and Isolated Lateralized Overgrowth.

OBJECTIVE: To provide information on evolution over time of leg length discrepancy in patients with syndromic and isolated lateralized overgrowth. STUDY DESIGN: This retrospective study investigates leg length discrepancy longitudinally in 105 patients with lateralized overgrowth either isolated (n = 37) or associated with Beckwith-Wiedemann spectrum (n = 56) or PIK3CA-related overgrowth spectrum (n = 12). Discrepancy was measured by standard methods and categorized as minor, mild, severe, and critical, based on the thresholds of 1, 2 and 5, respectively. RESULTS: The period of observation from diagnosis was 1.7 ± 2.6 to 9.0 ± 6.0 years. Leg length discrepancy was 11.0 ± 7.2 mm at diagnosis and 17.1 ± 14.4 mm at last visit. Both final leg length discrepancy and change over time were correlated with discrepancy at diagnosis (r2 = 0.45, P < .001 and r2 = 0.05, P = .019, respectively). Among minor leg length discrepancy at diagnosis, 47.5% remained minor, 40.0% become mild, and 12.5% severe. Among patients with discrepancy classified as severe at diagnosis, 84.6% remained severe and 15.4% evolved to critical. The isolated lateralized overgrowth group showed a milder evolution over time compared with Beckwith-Wiedemann spectrum and PIK3CA-related overgrowth spectrum groups. Among patients with Beckwith-Wiedemann, those with paternal chromosome 11 uniparental disomy had more severe leg length discrepancy at diagnosis and evolution over time. CONCLUSIONS: Leg length discrepancy associated with isolated or syndromic lateralized overgrowth tends to worsen with growth and correlates with discrepancy at first observation. Among the genotypic groups, isolated lateralized overgrowth tends to have a milder evolution, whereas Beckwith-Wiedemann spectrum predisposes to a more severe outcome, especially if associated with paternal chromosome 11 uniparental disomy genotype.

A child with complementary mosaic trisomy 8 and mosaic trisomy 21; clinical description of Warkany-Down syndrome and mechanism of origin.

Aneuploidy mosaicism involving two complementary different autosomal trisomy cell lines is extremely rare. Although a mosaic double trisomy 8/trisomy 21 has been described in literature, this is the first report of Warkany (+8)-Down (+21) syndrome due to two complementary mosaic trisomy cell lines. The phenotype of the male patient with Warkany-Down syndrome includes upslanting palpebral fissures, hypertelorism, small low-set ears with unilateral aural stenosis, large and broad hands and feet with deep palmar and plantar creases, bilateral cryptorchidism, generalized mild hypotonia and transient neonatal thrombocytopenia. At the age of two years, his developmental quotient is around 50. His height, weight and head circumference are below the third centile. We speculate on the mechanism of origin of the complementary trisomy cell lines based on molecular cytogenetic studies that showed no evidence for a chimera.

Inference of maternal uniparental disomy of the entire chromosome 2 from a paternity test.

Atypical situations arise during the constant resolution of paternity cases, which constitute challenges requiring additional genetic systems and non-standard methods. We report a paternity case presenting three alleged father (AF)-child incompatibilities for the markers TPOX, D2S441, and the indel locus B02 (11/11 vs 8/8; 14/14 vs 10/10; 2/2 vs1/1, respectively). Considering the presence of mutations/null alleles, the residual paternity indexes (PI) obtained with 23 autosomal short tandem repeats (STRs) and 38 indels suggest that the AF is the father (PI = 1.94e+011). Although the presence of few incompatibilities also could imply paternity of the AF brother, this hypothesis was less probable (PI = 3.20e+9) (W = 98.4 vs 1.6%, respectively). The inclusion of 23 Y-STR loci confirmed the paternity relationship in this case (global PI = 6.08e+15). However, the two multistep STRs and one indel incompatibilities allow discarding the mutation possibility. On the other hand, the confirmation of the homozygous STR genotypes with two different human identification kits and the low probability to find three null alleles (3.10e-8) allow rejecting the null allele presence hypothesis. Conversely, the child's homozygous genotype for maternal alleles in four markers located in the p and q arms of the chromosome 2 (TPOX, D2S441, D2S1338, and B02) suggests that maternal uniparental isodisomy better explains the relationship despite the presence of three paternal incompatibilities. In brief, when multiple incompatibilities are observed in paternity testing, the chromosomal location of the excluding loci and the use of additional genetic systems can be crucial to get confident kinship conclusions.

Insight into the mechanisms and consequences of recurrent telomere capture associated with a sub-telomeric deletion.

A complex mosaicism of the short arm of chromosome 1 detected by SNP microarray analysis is described in a patient presenting a 4-Mb 1p36 terminal deletion and associated phenotypic features. The array pattern of chromosome 1p displayed an intriguing increase in divergence of the SNP heterozygote frequency from the expected 50% from the centromere towards the 1p36 breakpoint. This suggests that various overlapping segments of UPD were derived by somatic recombination between the 1p homologues. The most likely explanation was the occurrence of a series of events initiated in either a gamete or an early embryonic cell division involving a 1pter deletion rapidly followed by multiple telomere captures, resulting in additive, stepped increases in frequency of homozygosity towards the telomere. The largest segment involved the entire 1p, and at least four other capture events were observed, indicating that at least five independent telomere captures occurred in separate cell lineages. The determination of breakpoint position by detection of abrupt changes in B-allele frequency using a moving window analysis demonstrated that they were identical in blood and saliva, the tissues available for analysis. We developed a model to explain the interaction of parameters determining the mosaic clones and concluded that, while number, size, and position of telomere captures were important initiating determinants, variation in individual clone frequencies was the main contributor to mosaic differences between tissues. All previous reports of telomere capture have been restricted to single events. Other cases involving multiple telomere capture probably exist but require investigation by SNP microarrays for their detection.

Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13 / Variation analysis of the number of copies and methylene patterns in region 15q11-q13

La región q11-q13 del cromosoma 15 humano es proclive a sufrir alteraciones genéticas. Algunos genes de la región presentan expresión parental diferencial monoalélica, regulada por imprinting (EI). Errores en la regulación del EI, disomías uniparentales (DSU), así como también el cambio en el número de copias genómicas (CNV) producidos por sitios susceptibles de quiebre cromosómico (BP), producen alteraciones en esta región. Las enfermedades más frecuentes asociadas son el síndrome de Prader-Willi, el síndrome de Angelman y el síndrome de microduplicación 15q11-q13. En el presente trabajo analizamos la región 15q11-q13 por Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA) en 181 muestras de ADN derivadas a nuestro servicio de análisis genético molecular. En este trabajo mostramos que, de las 181 muestras, 39 presentaron alteraciones detectables por MS-MLPA. El 61.5% (24/39) de esas alteraciones detectadas fueron deleciones, el 5.1% (2/39) duplicaciones y el 33.3%(13/39) DSU/EI. Los CNV fueron 4 veces más frecuentes que las DSU/EI (OR = 4; IC 95%: 1.56-10.25) consistente con la literatura. Entre los CNV, dos casos atípicos permiten postular posibles sitios BP que no han sido informados en la literatura previamente.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

[Variation analysis of the number of copies and methylene patterns in region 15q11-q13]. / Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

Single-nucleotide polymorphism array (SNP-A) improves the identification of chromosomal abnormalities by metaphase cytogenetics in myelodysplastic syndrome.

AIMS: The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell disorders characterised by inefficient haematopoiesis and risk of progression to acute myeloid leukaemia. Metaphase cytogenetics is an extremely valuable clinical tool in the management of haematological malignancies. However, metaphase cytogenetics requires cellular proliferation, its sensitivity and resolution depends on the proportion of clonal cells in the sample and size of the lesion, respectively. Single-nucleotide polymorphism array (SNP-A) does not depend on the presence of dividing cells, is able to detect copy number variations with a high resolution and to detect copy number neutral loss of heterozygosity or uniparental disomy (UPD). The aim of this study was to illustrate that the use of SNP-A can cover cryptic chromosomal lesions not identified by metaphase cytogenetics in patients with MDS. METHODS: Metaphase cytogenetics was performed on bone marrow aspirate using standard methods. Genomic DNA from total bone marrow cells were submitted to SNP-A using Affymetrix Genome-Wide Human SNP CytoScan HD. RESULTS: In our cohort of 15 patients with a diagnosis of MDS and related diseases, chromosomal abnormalities were found in 47% of the cases by SNP-A and in 33% by metaphase cytogenetics. SNP-A detected all lesions identified by metaphase cytogenetics, except a balanced translocation and a marker chromosome. Notably, SNP-A detected a total of 30 new lesions: 1 (3%) gain, 17 (57%) losses and 12 (40%) UPDs in 5 patients with MDS. CONCLUSIONS: SNP-A may complement metaphase cytogenetics to improve the detection of chromosomal abnormalities in myeloid neoplasms.

Do you know this syndrome? Dyspigmentation along the Blaschko lines caused by trisomy 7 mosaicism.

Dyspigmentation along the Blaschko lines is strongly suggestive of a mosaic skin disorder. We report a 9-year-old male patient who presented with swirls and streaks of both hypo and hyperpigmentation involving the entire body. Additionally, he had hypertrichosis, musculoskeletal and minor neurodevelopment abnormalities but no intellectual disability. Cultured fibroblast displayed trisomy 7 mosaicism, which can explain this pigmentary phenotype. Widespread dyspigmentation associated with involvement of other organs should prompt systemic examination to detect additional anomalies and genetic evaluation should be considered, even with normal fetal karyotype.

Absence of heterozygosity due to template switching during replicative rearrangements.

We investigated complex genomic rearrangements (CGRs) consisting of triplication copy-number variants (CNVs) that were accompanied by extended regions of copy-number-neutral absence of heterozygosity (AOH) in subjects with multiple congenital abnormalities. Molecular analyses provided observational evidence that in humans, post-zygotically generated CGRs can lead to regional uniparental disomy (UPD) due to template switches between homologs versus sister chromatids by using microhomology to prime DNA replication-a prediction of the replicative repair model, MMBIR. Our findings suggest that replication-based mechanisms might underlie the formation of diverse types of genomic alterations (CGRs and AOH) implicated in constitutional disorders.

Angelman syndrome in adulthood.

Angelman syndrome (AS) is a neurogenetic disorder. The goal of this study was to investigate the primary health issues affecting adults with AS and to further characterize the natural history and genotype-phenotype correlations. Standardized phone interviews with caregivers for 110 adolescents and adults with AS were conducted. The impact of age, sex, and genotype on specific outcomes in neurology, orthopedics, internal medicine, and psychiatry were investigated. The mean age of individuals with AS was 24 years (range 16-50y). Active seizures were present in 41% of individuals, and 72% had sleep dysfunction. Significant constipation was present in 85%, and 32% were overweight or obese, with obesity disproportionately affecting women. Scoliosis affected 50% with a mean age at diagnosis of 12 years, and 24% of those diagnosed with scoliosis required surgery, an intervention disproportionately affecting men. Sixty-eight percent were able to walk independently, and 13% were able to speak 5 or more words. Self-injurious behavior was exhibited in 52% of individuals. The results of this study indicate that epilepsy severity may assume a bimodal age distribution: seizures are typically most severe in early childhood but may recur in adulthood. While late-adolescent and adult sleep patterns were improved when compared to the degree of sleep dysfunction present during infancy and childhood, the prevalence of poor sleep in adults remained quite high. Primary areas of clinical management identified include the following: seizures, sleep, aspiration risk, GERD, constipation, dental care, vision, obesity, scoliosis, bone density, mobility, communication, behavior, and anxiety.

Segmental uniparental isodisomy of chromosome 6 causing transient diabetes mellitus and merosin-deficient congenital muscular dystrophy.

Segmental uniparental isodisomy (iUPD) is a rare genetic event that may cause aberrant expression of imprinted genes, and reduction to homozygosity of a recessive mutation. Transient neonatal diabetes mellitus (TNDM) is typically caused by imprinting aberrations in chromosome 6q24 TNDM differentially-methylated region (DMR). Approximately, 15.12 Mb upstream in 6q22-q23 is located LAMA2, the gene responsible of merosin-deficient congenital muscular dystrophy type 1A (MDC1A). We investigated a patient diagnosed both with TNDM and MDC1A, born from a twin dichorionic discordant pregnancy. Parents are first-degree cousins. Methylation sensitive-PCR of the imprinted 6q24 TNDM CpG island showed only the non-methylated (paternal) allele. Microsatellite markers and SNP array profiling disclosed normal biparental inheritance at 6p and a segmental paternal iUPD, between 6q22.33 and 6q27. Sequencing of LAMA2 exons showed a homozygous frameshift mutation, c.7490_7493dupAAGA, which predicts p.Asp2498GlufsX4, in exon 54. Her father, but not her mother, was a carrier of the mutation. While segmental paternal iUPD6 causing TNDM was reported twice, there are no previous reports of MDC1A caused by this event. This is a child with two genetic disorders, yet neither is caused by the parental consanguinity, which reinforces the importance of considering different etiological mechanisms in the genetic clinic.