Identification of Pathogenic Copy Number Variants in Mexican Patients With Inherited Retinal Dystrophies Applying an Exome Sequencing Data-Based Read-Depth Approach.

BACKGROUND: Retinal dystrophies (RDs) are the most common cause of inherited blindness worldwide and are caused by genetic defects in about 300 different genes. While targeted next-generation sequencing (NGS) has been demonstrated to be a reliable and efficient method to identify RD disease-causing variants, it doesn't routinely identify pathogenic structural variant as copy number variations (CNVs). Targeted NGS-based CNV detection has become a crucial step for RDs molecular diagnosis, particularly in cases without identified causative single nucleotide or Indels variants. Herein, we report the exome sequencing (ES) data-based read-depth bioinformatic analysis in a group of 30 unrelated Mexican RD patients with a negative or inconclusive genetic result after ES. METHODS: CNV detection was performed using ExomeDepth software, an R package designed to detect CNVs using exome data. Bioinformatic validation of identified CNVs was conducted through a commercially available CNV caller. All identified candidate pathogenic CNVs were orthogonally verified through quantitative PCR assays. RESULTS: Pathogenic or likely pathogenic CNVs were identified in 6 out of 30 cases (20%), and of them, a definitive molecular diagnosis was reached in 5 cases, for a final diagnostic rate of ~17%. CNV-carrying genes included CLN3 (2 cases), ABCA4 (novel deletion), EYS, and RPGRIP1. CONCLUSIONS: Our results indicate that bioinformatic analysis of ES data is a reliable method for pathogenic CNV detection and that it should be incorporated in cases with a negative or inconclusive molecular result after ES.

Identification of mutations in canine oral mucosal melanomas by exome sequencing and comparison with human melanomas.

Oral mucosal melanomas (OMMs) are aggressive neoplasms commonly found in dogs but rare in humans. Utilizing whole exome sequencing (WES), which focuses on protein-coding regions to reveal mutation profiles, we conducted a comparative analysis of canine OMM and human melanomas. This study involved DNA extraction, exome enrichment, and sequencing from three canine OMM cell lines (CMGD-2, CMGD-5, TLM-1), five canine OMM frozen samples, a human OMM cell line (MEMO), and a human commercial skin melanoma cell line (SK-MEL-28). The sequencing and subsequent analysis of FASTQ files yielded final variant files, leading to the identification of mutations. Our findings revealed a total of 500 mutated genes in canine OMM, including significant ones such as EP300, FAT4, JAK3, LRP1B, NCOR1, and NOTCH1. Notably, 82 shared mutations were identified between human melanomas and canine OMM genomes. These mutations were categorized based on the gene functions. The identification of these mutations provides critical insights that can pave the way for the development of novel therapeutic strategies for both canine and human OMM, offering hope for more effective treatments in the future.

Identification and functional characteristics of CHD1L gene variants implicated in human Müllerian duct anomalies.

BACKGROUND: Müllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations. METHODS: Our previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays. RESULTS: Ultimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs. CONCLUSIONS: The current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.

Deciphering genetic and nongenetic factors underlying tumour dormancy: insights from multiomics analysis of two syngeneic MRD models of melanoma and leukemia.

BACKGROUND: Tumour dormancy, a resistance mechanism employed by cancer cells, is a significant challenge in cancer treatment, contributing to minimal residual disease (MRD) and potential relapse. Despite its clinical importance, the mechanisms underlying tumour dormancy and MRD remain unclear. In this study, we employed two syngeneic murine models of myeloid leukemia and melanoma to investigate the genetic, epigenetic, transcriptomic and protein signatures associated with tumour dormancy. We used a multiomics approach to elucidate the molecular mechanisms driving MRD and identify potential therapeutic targets. RESULTS: We conducted an in-depth omics analysis encompassing whole-exome sequencing (WES), copy number variation (CNV) analysis, chromatin immunoprecipitation followed by sequencing (ChIP-seq), transcriptome and proteome investigations. WES analysis revealed a modest overlap of gene mutations between melanoma and leukemia dormancy models, with a significant number of mutated genes found exclusively in dormant cells. These exclusive genetic signatures suggest selective pressure during MRD, potentially conferring resistance to the microenvironment or therapies. CNV, histone marks and transcriptomic gene expression signatures combined with Gene Ontology (GO) enrichment analysis highlighted the potential functional roles of the mutated genes, providing insights into the pathways associated with MRD. In addition, we compared "murine MRD genes" profiles to the corresponding human disease through public datasets and highlighted common features according to disease progression. Proteomic analysis combined with multi-omics genetic investigations, revealed a dysregulated proteins signature in dormant cells with minimal genetic mechanism involvement. Pathway enrichment analysis revealed the metabolic, differentiation and cytoskeletal remodeling processes involved in MRD. Finally, we identified 11 common proteins differentially expressed in dormant cells from both pathologies. CONCLUSIONS: Our study underscores the complexity of tumour dormancy, implicating both genetic and nongenetic factors. By comparing genomic, transcriptomic, proteomic, and epigenomic datasets, our study provides a comprehensive understanding of the molecular landscape of minimal residual disease. These results provide a robust foundation for forthcoming investigations and offer potential avenues for the advancement of targeted MRD therapies in leukemia and melanoma patients, emphasizing the importance of considering both genetic and nongenetic factors in treatment strategies.

Principles of clinical genetics for rheumatologists: clinical indications and interpretation of broad-based genetic testing.

Advances in DNA sequencing technologies, especially next-generation sequencing (NGS), which is the basis for whole-exome sequencing (WES) and whole-genome sequencing (WGS), have profoundly transformed immune-mediated rheumatic disease diagnosis. Recently, substantial cost reductions have facilitated access to these diagnostic tools, expanded the capacity of molecular diagnostics and enabled the pursuit of precision medicine in rheumatology. Understanding the fundamental principles of genetics and diversity in genetic variant classification is a crucial milestone in rheumatology. However, despite the growing availability of DNA sequencing platforms, a significant number of autoinflammatory diseases (AIDs), neuromuscular disorders, hereditary collagen diseases, and monogenic bone diseases remain unsolved, and variants of uncertain significance (VUS) pose a formidable challenge to addressing these unmet needs in the coming decades. This article aims to provide an overview of the clinical indications and interpretation of comprehensive genetic testing in the medical field, addressing the related complexities and implications.

Deciphering the etiology of undiagnosed ocular anomalies along with systemic alterations in pediatric patients through whole exome sequencing.

Inherited and developmental eye diseases are quite diverse and numerous, and determining their genetic cause is challenging due to their high allelic and locus heterogeneity. New molecular approaches, such as whole exome sequencing (WES), have proven to be powerful molecular tools for addressing these cases. The present study used WES to identify the genetic etiology in ten unrelated Mexican pediatric patients with complex ocular anomalies and other systemic alterations of unknown etiology. The WES approach allowed us to identify five clinically relevant variants in the GZF1, NFIX, TRRAP, FGFR2 and PAX2 genes associated with Larsen, Malan, developmental delay with or without dysmorphic facies and autism, LADD1 and papillorenal syndromes. Mutations located in GZF1 and NFIX were classified as pathogenic, those in TRRAP and FGFR2 were classified as likely pathogenic variants, and those in PAX2 were classified as variants of unknown significance. Protein modeling of the two missense FGFR2 p.(Arg210Gln) and PAX2 p.(Met3Thr) variants showed that these changes could induce potential structural alterations in important functional regions of the proteins. Notably, four out of the five variants were not previously reported, except for the TRRAP gene. Consequently, WES enabled the identification of the genetic cause in 40% of the cases reported. All the syndromes reported herein are very rare, with phenotypes that may overlap with other genetic entities.

Germline mutations of breast cancer susceptibility genes through expanded genetic analysis in unselected Colombian patients.

BACKGROUND: In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted. RESULTS: We ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure. CONCLUSION: This study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.

FLNC Associated Restrictive Cardiomyopathy and Hypertrabeculation, a Rare Association. / FLNC Associada a Cardiomiopatia Restritiva e Hipertrabeculação, uma Associação Rara.

A six-year-old girl with restrictive cardiomyopathy and hypertrabeculation, due to the early onset of her disease, whole exome sequencing was conducted, revealing the presence of a novel heterozygous missense variant in the FLNC gene. The same gene variant was also identified in her father, who, at an adult age, displayed normal imaging results and was symptom-free. This variant has not been reported in population databases or current medical literature and is classified as likely pathogenic.

Menina de seis anos com cardiomiopatia restritiva e hipertrabeculação na qual, devido ao início precoce da doença, foi realizado sequenciamento completo do exoma, revelando a presença de uma nova variante heterozigótica missense no gene FLNC. A mesma variante genética também foi identificada em seu pai, que, já adulto, apresentava resultados de imagem normais e não apresentava sintomas. Esta variante não foi relatada em bancos de dados populacionais ou na literatura médica atual e é classificada como provavelmente patogênica.

Knockout mice with pituitary malformations help identify human cases of hypopituitarism.

BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.

Comprehensive genomic filtering algorithm to expose the cause of skewed X chromosome inactivation. The proof of concept in female haemophilia expression.

BACKGROUND: Exploring the expression of X linked disorders like haemophilia A (HA) in females involves understanding the balance achieved through X chromosome inactivation (XCI). Skewed XCI (SXCI) may be involved in symptomatic HA carriers. We aimed to develop an approach for dissecting the specific cause of SXCI and verify its value in HA. METHODS: A family involving three females (two symptomatic with severe/moderate HA: I.2, the mother, and II.1, the daughter; one asymptomatic: II.2) and two related affected males (I.1, the father and I.3, the maternal uncle) was studied. The genetic analysis included F8 mutational screening, multiplex ligation-dependent probe amplification, SNP microarray, whole exome sequencing (WES) and Sanger sequencing. XCI patterns were assessed in ectoderm/endoderm and mesoderm-derived tissues using AR-based and RP2-based systems. RESULTS: The comprehensive family analysis identifies I.2 female patient as a heterozygous carrier of F8:p.(Ser1414Ter) excluding copy number variations. A consistent XCI pattern of 99.5% across various tissues was observed. A comprehensive filtering algorithm for WES data was designed, developed and applied to I.2. A Gly58Arg missense variant in VMA21 was revealed as the cause for SXCI.Each step of the variant filtering system takes advantage of publicly available genomic databases, non-SXCI controls and case-specific molecular data, and aligns with established concepts in the theoretical background of SXCI. CONCLUSION: This study acts as a proof of concept for our genomic filtering algorithm's clinical utility in analysing X linked disorders. Our findings clarify the molecular aspects of SXCI and improve genetic diagnostics and counselling for families with X linked diseases like HA.

Neurodevelopmental disorder associated with gene ARF3: A case report.

We present a case study of a patient exhibiting acquired microcephaly along with global developmental delay and drug-resistant epilepsy. Brain magnetic resonance imaging revealed distinctive features, including a Z-shaped morphology of the brainstem, volumetric reduction of white matter, diffuse thinning of the corpus callosum, and partial fusion of the cerebellar hemispheres at their most cranial portion. Whole-exome sequencing uncovered a pathogenic variant in the ARF3 gene c.200A>T, p.(Asp67Val). The neurodevelopmental disorder associated with the ARF3 gene is exceptionally rare, with only two previously documented cases in the literature. This disorder is characterized by global developmental delay and brain malformations, particularly affecting the white matter, cerebellum, and brainstem. It can also manifest as acquired microcephaly and epilepsy. These phenotypic characteristics align with Golgipathies, underscoring the significance of considering this group of conditions in relevant clinical contexts. In cases where a Z-shaped morphology of the brainstem is observed, ARF3-associated disorder should be included in the list of differential diagnoses.

[Multiple endocrine neoplasia and very early onset inflammatory bowel disease. An unexpected association]. / Neoplasia endócrina múltiple y enfermedad inflamatoria intestinal de inicio muy temprano. Una asociación inesperada.

Very early onset inflammatory bowel disease (VEOIBD) is a rare entity in pediatrics. Its association with primary immunodeficiencies of monogenic origin is known. We present the case of a patient diagnosed with VEOIBD who underwent massive paralleled exome sequencing. The result of the study showed a pathogenic variant in the RET proto-oncogene, associated with multiple endocrine neoplasia type 2A disease. There are no previous reports of association of RET proto-oncogene variants with VEOIBD. The presence of these two clinical entities cannot be attributed to a single genetic cause.

La enfermedad inflamatoria intestinal de inicio muy temprano (VEOIBD) es una entidad rara en pediatría. Es conocida su asociación con inmunodeficiencias primarias de origen monogénico. Presentamos el caso de una paciente con diagnóstico de VEOIBD a quien se le realizó una secuenciación masiva del exoma. El resultado del estudio permitió identificar una variante patogénica en el proto oncogen RET, asociada con enfermedad neoplasia endocrina múltiple tipo 2A. No hay reportes de asociación de variantes en el proto oncogen RET con VEOIBD. No se puede adjudicar la presencia de estas dos entidades clínicas a una única causa genética.

Copy number signatures and CCNE1 amplification reveal the involvement of replication stress in high-grade endometrial tumors oncogenesis.

PURPOSE: Managing high-grade endometrial cancer in Martinique poses significant challenges. The diversity of copy number alterations in high-grade endometrial tumors, often associated with a TP53 mutation, is a key factor complicating treatment. Due to the high incidence of high-grade tumors with poor prognosis, our study aimed to characterize the molecular signature of these tumors within a cohort of 25 high-grade endometrial cases. METHODS: We conducted a comprehensive pangenomic analysis to categorize the copy number alterations involved in these tumors. Whole-Exome Sequencing (WES) and Homologous Recombination (HR) analysis were performed. The alterations obtained from the WES were classified into various signatures using the Copy Number Signatures tool available in COSMIC. RESULTS: We identified several signatures that correlated with tumor stage and disctinct prognoses. These signatures all seem to be linked to replication stress, with CCNE1 amplification identified as the primary driver of oncogenesis in over 70% of tumors analyzed. CONCLUSION: The identification of CCNE1 amplification, which is currently being explored as a therapeutic target in clinical trials, suggests new treatment strategies for high-grade endometrial cancer. This finding holds particular significance for Martinique, where access to care is challenging.

Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

PURPOSE: In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. METHODS: A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. RESULTS: The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. CONCLUSIONS: Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

Advantages of whole-exome sequencing over immunomapping in 67 Brazilian patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is characterized by skin fragility and blistering. In Brazil, the diagnosis is usually obtained through immunomapping, which involves a skin biopsy. Most recently, whole exome sequencing (WES) has become an important tool for the diagnosis of the subtypes of EB, providing information on prognosis as well as allowing appropriate genetic counseling for the families. OBJECTIVE: To compare the results of immunomapping and molecular analysis and to describe the characteristics of a Brazilian cohort of patients with EB. METHODS: Patients were submitted to clinical evaluation and WES using peripheral blood samples. WES results were compared to those obtained from immunomapping testing from skin biopsies. RESULTS: 67 patients from 60 families were classified: 47 patients with recessive dystrophic EB (DEB), 4 with dominant DEB, 15 with EB simplex (EBS), and 1 with junctional EB (JEB). Novel causative variants were: 10/60 (16%) in COL7A1 associated with recessive DEB and 3 other variants in dominant DEB; one homozygous variant in KRT5 and another homozygous variant in PLEC, both associated with EBS. Immunomapping was available for 59 of the 67 patients and the results were concordant with exome results in 37 (62%), discordant in 13 (22%), and inconclusive in 9 patients (15%). STUDY LIMITATIONS: Even though EB is a rare disease, for statistical purposes, the number of patients evaluated by this cohort can still be considered limited; other than that, there was a significant difference between the proportion of types of EB (only one case with JEB, against more than 50 with DEB), which unfortunately represents a selection bias. Also, for a small subset of families, segregation (usually through Sanger sequencing) was not an option, usually due to deceased or unknown parent status (mostly the father). CONCLUSION: Although immunomapping has been useful in services where molecular studies are not available, this invasive method may provide a misdiagnosis or an inconclusive result in about 1/3 of the patients. This study shows that WES is an effective method for the diagnosis and genetic counseling of EB patients.

RET 634 germline/gonadal mosaicism generating a second pathogenic amino acid change in multiple endocrine neoplasia type 2A.

Genetic testing for germline RET pathogenic variants, which cause the Multiple Endocrine Neoplasia Type 2 (MEN2) syndrome, has become crucial in managing patients with medullary thyroid carcinoma (MTC). Classically, RET heterozygous missense pathogenic variants are transmitted in a Mendelian autosomal dominant pattern, of which germline/gonadal mosaicism has never been reported. We report the novel occurrence of a MEN2A patient's family in which the siblings inherited three different RET 634 genotypes: wild type (p.Cys634), p.Cys634Gly or p.Cys634Arg heterozygous pathogenic variants. We hypothesized that germline/gonadal mosaicism, derived from an inherited + early somatic mutation in the mother or a double de novo mutation during maternal embryogenesis, led to this rare event in the RET gene. Exome analysis of the proband's deceased mother's paraffin-embedded thyroid tissue confirmed the three nucleotides in the same 634 codon position. For the first time, we describe germline/gonadal mosaicism in RET, generating a second pathogenic amino acid change in the same codon causing MEN2A. Our finding shows that RET parental mosaicism, confirmed by somatic exome sequencing, might explain discrepant genotype cases in siblings with inherited cancers.

Clinical Characterization and Underlying Genetic Findings in Brazilian Patients with Syndromic Microcephaly Associated with Neurodevelopmental Disorders.

Microcephaly is characterized by an occipitofrontal circumference at least two standard deviations below the mean for age and sex. Neurodevelopmental disorders (NDD) are commonly associated with microcephaly, due to perturbations in brain development and functioning. Given the extensive genetic heterogeneity of microcephaly, managing patients is hindered by the broad spectrum of diagnostic possibilities that exist before conducting molecular testing. We investigated the genetic basis of syndromic microcephaly accompanied by NDD in a Brazilian cohort of 45 individuals and characterized associated clinical features, as well as evaluated the effectiveness of whole-exome sequencing (WES) as a diagnostic tool for this condition. Patients previously negative for pathogenic copy number variants underwent WES, which was performed using a trio approach for isolated index cases (n = 31), only the index in isolated cases with parental consanguinity (n = 8) or affected siblings in familial cases (n = 3). Pathogenic/likely pathogenic variants were identified in 19 families (18 genes) with a diagnostic yield of approximately 45%. Nearly 86% of the individuals had global developmental delay/intellectual disability and 51% presented with behavioral disturbances. Additional frequent clinical features included facial dysmorphisms (80%), brain malformations (67%), musculoskeletal (71%) or cardiovascular (47%) defects, and short stature (54%). Our findings unraveled the underlying genetic basis of microcephaly in half of the patients, demonstrating a high diagnostic yield of WES for microcephaly and reinforcing its genetic heterogeneity. We expanded the phenotypic spectrum associated with the condition and identified a potentially novel gene (CCDC17) for congenital microcephaly.

Decoding complex inherited phenotypes in rare disorders: the DECIPHERD initiative for rare undiagnosed diseases in Chile.

Rare diseases affect millions of people worldwide, and most have a genetic etiology. The incorporation of next-generation sequencing into clinical settings, particularly exome and genome sequencing, has resulted in an unprecedented improvement in diagnosis and discovery in the past decade. Nevertheless, these tools are unavailable in many countries, increasing health care gaps between high- and low-and-middle-income countries and prolonging the "diagnostic odyssey" for patients. To advance genomic diagnoses in a setting of limited genomic resources, we developed DECIPHERD, an undiagnosed diseases program in Chile. DECIPHERD was implemented in two phases: training and local development. The training phase relied on international collaboration with Baylor College of Medicine, and the local development was structured as a hybrid model, where clinical and bioinformatics analysis were performed in-house and sequencing outsourced abroad, due to lack of high-throughput equipment in Chile. We describe the implementation process and findings of the first 103 patients. They had heterogeneous phenotypes, including congenital anomalies, intellectual disabilities and/or immune system dysfunction. Patients underwent clinical exome or research exome sequencing, as solo cases or with parents using a trio design. We identified pathogenic, likely pathogenic or variants of unknown significance in genes related to the patients´ phenotypes in 47 (45.6%) of them. Half were de novo informative variants, and half of the identified variants have not been previously reported in public databases. DECIPHERD ended the diagnostic odyssey for many participants. This hybrid strategy may be useful for settings of similarly limited genomic resources and lead to discoveries in understudied populations.

Identification of Genetic Variants for Diabetic Retinopathy Risk Applying Exome Sequencing in Extreme Phenotypes.

Background: Diabetic retinopathy (DR) risk has been shown to vary depending on ethnic backgrounds, and thus, it is worthy that underrepresented populations are analyzed for the potential identification of DR-associated genetic variants. We conducted a case-control study for the identification of DR-risk variants in Mexican population. Methods: We ascertained 60 type 2 diabetes mellitus (T2DM) patients. Cases (n = 30) were patients with advanced proliferative DR (PDR) with less than 15 years after a T2DM diagnosis while controls (n = 30) were patients with no DR 15 years after the diagnosis of T2DM. Exome sequencing was performed in all patients, and the frequency of rare variants was compared. In addition, the frequency of variants occurring in a set of 169 DR-associated genes were compared. Results: Statistically significant differences were identified for rare missense and splice variants and for rare splice variants occurring more than once in either group. A strong statistical difference was observed when the number of rare missense variants with an aggregated prediction of pathogenicity and occurring more than once in either group was compared (p = 0.0035). Moreover, 8 variants identified more than once in either group, occurring in previously identified DR-associated genes were recognized. The p.Pro234Ser KIR2DS4 variant showed a strong protective effect (OR = 0.04 [0.001-0.36]; p = 0.04). Conclusions: Our study showed an enrichment of rare splice acceptor/donor variants in patients with PDR and identified a potential protective variant in KIR2DS4. Although statistical significance was not reached, our results support the replication of 8 previously identified DR-associated genes.

Germline mutations in pediatric cancer cohort with mixed-ancestry Mexicans.

BACKGROUND: Childhood cancer is one of the primary causes of disease-related death in 5- to 14-year-old children and currently no prevention strategies exist to reduce the incidence of this disease. Childhood cancer has a larger hereditary component compared with cancer in adults. Few genetic studies have been conducted on children with cancer. Additionally, Latin American populations are underrepresented in genomic studies compared with other populations. Therefore, the aim of this study is to analyze germline mutations in a group of mixed-ancestry Mexican pediatric patients with solid and hematological cancers. METHODS: We analyzed genetic variants from 40 Mexican childhood cancer patients and their relatives. DNA from saliva or blood samples was used for whole-exome sequencing. All variants were identified following GATK best practices. RESULTS: We found that six patients (15%) were carriers of germline mutations in CDKN2A, CHEK2, DICER1, FANCA, MSH6, MUTYH, NF1, and SBDS cancer predisposition genes, and additional new variants predicted to be deleterious by in silico algorithms. A population genetics analysis detected five components consistent with the demographic models assumed for modern mixed-ancestry Mexicans. CONCLUSIONS: This report identifies potential genetic risk factors and provides a better understanding of the underlying mechanisms of childhood cancer in this population.