Functional Insight into and Refinement of the Genomic Boundaries of the JARID2-Neurodevelopmental Disorder Episignature.

JARID2 (Jumonji, AT-rich interactive domain 2) haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome. It is characterized by intellectual disability, developmental delay, autistic features, behavior abnormalities, cognitive impairment, hypotonia, and dysmorphic features. JARID2 acts as a transcriptional repressor protein that is involved in the regulation of histone methyltransferase complexes. JARID2 plays a role in the epigenetic machinery, and the associated syndrome has an identified DNA methylation episignature derived from sequence variants and intragenic deletions involving JARID2. For this study, our aim was to determine whether patients with larger deletions spanning beyond JARID2 present a similar DNA methylation episignature and to define the critical region involved in aberrant DNA methylation in 6p22-p24 microdeletions. We examined the DNA methylation profiles of peripheral blood from 56 control subjects, 13 patients with (likely) pathogenic JARID2 variants or patients carrying copy number variants, and three patients with JARID2 VUS variants. The analysis showed a distinct and strong differentiation between patients with (likely) pathogenic variants, both sequence and copy number, and controls. Using the identified episignature, we developed a binary model to classify patients with the JARID2-neurodevelopmental syndrome. DNA methylation analysis indicated that JARID2 is the driver gene for aberrant DNA methylation observed in 6p22-p24 microdeletions. In addition, we performed analysis of functional correlation of the JARID2 genome-wide methylation profile with the DNA methylation profiles of 56 additional neurodevelopmental disorders. To conclude, we refined the critical region for the presence of the JARID2 episignature in 6p22-p24 microdeletions and provide insight into the functional changes in the epigenome observed when regulation by JARID2 is lost.

Contribution of Dynamic and Genetic Tests for Short Stature Diagnosing: A Case Report.

Genetic tests have led to the discovery of many novel genetic variants related to growth failure, but the clinical significance of some results is not always easy to establish. The aim of this report is to describe both clinical phenotype and genetic characteristics in an adult patient with short stature associated with a homozygous variant in disintegrin and metalloproteinase with thrombospondin motifs type 17 gene (ADAMTS17) combined with a homozygous variant in the GH secretagogue receptor (GHS-R). The index case had severe short stature (SS) (-3.0 SD), small hands and feet, associated with eye disturbances. Genetic tests revealed homozygous compounds for ADAMTS17 responsible for Weill-Marchesani-like syndrome but a homozygous variant in GHS-R was also detected. Dynamic stimulation with an insulin tolerance test showed a normal elevation of GH, while the GH response to macimorelin stimulus was totally flattened. We show the implication of the GHS-R variant and review the molecular mechanisms of both entities. These results allowed us to better interpret the phenotypic spectrum, associated co-morbidities, its implications in dynamic tests, genetic counselling and treatment options not only to the index case but also for her relatives.

An spanish study of secondary findings in families affected with mendelian disorders: choices, prevalence and family history.

Clinical exome sequencing has the potential to identify pathogenic variants unrelated to the purpose of the study (secondary findings, SFs). Data describing actual choices of SFs in participants in a clinical setting and factors influencing their decision are virtually non-existant in Europe. In this work, we report the acceptance rate of SFs, calculate their prevalence and study factors associated with the decision in a cohort of patients affected with a rare genetic disorder in a Spanish Hospital. Finally, we re-examine the presence of previously non reported family history in positive cases. We retrospectively reviewed informed consent choices and SF results from 824 unrelated probands affected with rare genetic disorders who underwent whole-genome or exome sequencing. Ninety percent of families (740/824) affected with rare disorders wished to be informed of SFs. Declining SFs was associated with a prenatal setting (30% vs. 8.7%, p = 0.025), consanguinity (19% vs. 8.7%, p = 0.013), male gender (10.6% vs. 1.5%, p = 0.00865) and the proband being a minor (10.6% vs. 1.5%, p = 0.014). Overall, 27 pathogenic or likely pathogenic variants were identified in 27 individuals, with an SF prevalence of 3.6%. Disclosure of SFs increased the percentage of positive family histories and resulted in early diagnosis or changes in the management of 10 individuals from five families. We show that the acceptance of SFs in Spain is high and the disclosure of SFs leads to a clinically meaningful change in the medical management of individuals.

Next-Generation Sequencing Gene Panels and "Solo" Clinical Exome Sequencing Applied in Structurally Abnormal Fetuses.

OBJECTIVE: The aim of the study was to assess the diagnostic yield of 2 different next-generation sequencing (NGS) approaches: gene panel and "solo" clinical exome sequencing (solo-CES), in fetuses with structural anomalies and normal chromosomal microarray analysis (CMA), in the absence of a known familial mutation. METHODOLOGY: Gene panels encompassing from 2 to 140 genes, were applied mainly in persistent nuchal fold/fetal hydrops and in large hyperechogenic kidneys. Solo-CES, which entails sequencing the fetus alone and only interpreting the Online Mendelian Inheritance in Man genes, was performed in multisystem or recurrent structural anomalies. RESULTS: During the study period (2015-2020), 153 NGS studies were performed in 148 structurally abnormal fetuses with a normal CMA. The overall diagnostic yield accounted for 35% (53/153) of samples and 36% (53/148) of the fetuses. Diagnostic yield with the gene panels was 31% (15/49), similar to 37% (38/104) in solo-CES. CONCLUSIONS: A monogenic disease was established as the underlying cause in 35% of selected fetal structural anomalies by gene panels and solo-CES.

Histopathology of recurrent Steel syndrome in fetuses caused by novel variants of COL27A1 gene.

Steel syndrome (STLS) encompasses characteristic facies, dwarfness, irreducible bilateral hip and radial head dislocation, and carpal bone coalition due to COL27A1 mutations. Two consecutive pregnancies in a non-consanguineous couple were terminated because of severe fetal anomalies. Complete autopsies with microscopic exam were performed on both fetuses. Next-generation-based clinical exome sequencing was applied to the first fetus. Exome sequencing results, parental segregation, and affection of the second fetus were confirmed by Sanger sequencing. Both fetuses had signs consistent with STLS. Bilateral capitulum humeri absence explained radial head dislocation in STLS. Metaphyseal cartilage showed severe disorganization. Resting cartilage was hypercellular, organized in irregular nests limited by acellular matrix. Two variants in COL27A1 (c.2548G>A -p.Gly850Arg- and c.3249+1G> T) were found in both fetuses in compound heterozygosity with parental Mendelian segregation. This is the first report to include histology of STLS. The COL27A1 variants here described increase the number of mutations associated with STLS.

Heterozygous STUB1 mutation causes familial ataxia with cognitive affective syndrome (SCA48).

OBJECTIVE: To describe a new spinocerebellar ataxia (SCA48) characterized by early cerebellar cognitive-affective syndrome (CCAS) and late-onset SCA. METHODS: This is a descriptive study of a family that has been followed for more than a decade with periodic neurologic and neuropsychological examinations, MRI, brain SPECT perfusion, and genetic analysis. Whole exome sequencing was performed in 3 affected and 1 unaffected family member and subsequently validated by linkage analysis of chromosome 16p13.3. RESULTS: Six patients fully developed cognitive-affective and complete motor cerebellar syndrome associated with vermian and hemispheric cerebellar atrophy, suggesting a continuum from a dysexecutive syndrome slowly evolving to a complete and severe CCAS with late truncal ataxia. Three presymptomatic patients showed focal cerebellar atrophy in the vermian, paravermian, and the medial part of cerebellar lobes VI and VII, suggesting that cerebellar atrophy preceded the ataxia, and that the neurodegeneration begins in cerebellar areas related to cognition and emotion, spreading later to the whole cerebellum. Among the candidate variants, only the frameshift heterozygous c.823_824delCT STUB1 (p.L275Dfs*16) pathogenic variant cosegregated with the disease. The p.L275Dfs*16 heterozygous STUB1 pathogenic variant leads to neurodegeneration and atrophy in cognition- and emotion-related cerebellar areas and reinforces the importance of STUB1 in maintaining cognitive cerebellar function. CONCLUSIONS: We report a heterozygous STUB1 pathogenic genetic variant causing dominant cerebellar ataxia. Since recessive mutations in STUB1 gene have been previously associated with SCAR16, these findings suggest a previously undescribed SCA locus (SCA48; MIM# 618093).

New subtype of spinocerebellar ataxia with altered vertical eye movements mapping to chromosome 1p32.

IMPORTANCE: To provide clinical and genetic diagnoses for patients' conditions, it is important to identify and characterize the different subtypes of spinocerebellar ataxia (SCA). OBJECTIVE: To clinically and genetically characterize a Spanish kindred with pure SCA presenting with altered vertical eye movements. DESIGN Family study of ambulatory patients. Electro-oculographic and genetics studies were performed in 2 referral university centers. SETTING: Primary care institutional center in Spain. PARTICIPANTS: Thirty-six participants from a large Spanish kindred were clinically examined, and 33 family members were genetically examined. Detailed clinical data were obtained from 9 affected relatives. Two ataxic siblings and 2 asymptomatic family members were examined using an enhanced clinical protocol for a follow-up period of 7 years. MAIN OUTCOMES AND MEASURES: High-density genome-wide single-nucleotide polymorphism arrays, along with microsatellite analysis, and genetic linkage studies were performed. Whole-exome sequencing was used for 2 affected relatives. For most patients, the initial symptoms included falls, dysarthria, or clumsiness followed by a complete cerebellar syndrome. For all 9 affected relatives, we observed altered vertical eye movements, as initial ocular signs for 3 of them and for the 2 asymptomatic family members, all having inherited the risk haplotype. Neuroimaging showed isolated cerebellar atrophy. RESULTS: Initial genome-wide linkage analysis revealed suggestive linkage to chromosome 1p32. Multipoint analysis and haplotype reconstruction further traced this SCA locus to a 0.66-cM interval flanked by D1S200 and D1S2742 (z(max) = 6.539; P < .0001). The causative mutation was unidentified by exome sequencing. CONCLUSIONS AND RELEVANCE: We report a new subtype of SCA presenting in patients as slow progressing ataxia with altered vertical eye movements linked to a 11-megabase interval on 1p32. The Human Genome Nomenclature Committee has assigned this subtype of ataxia the designation SCA37.

Germline mutations in NF1 and BRCA1 in a family with neurofibromatosis type 1 and early-onset breast cancer.

Neurofibromatosis type 1 (NF1) is a common dominant autosomal disorder caused by mutations in the NF1 gene. The main manifestations of NF1 are café-au-lait spots, neurofibromas, intertriginous freckling, Lisch nodules, and malignancy, including peripheral nerve sheath tumors, central nervous system gliomas, and a variety of other tumors not so clearly defined. The association between NF1 and breast cancer or other gynecologic malignancies seems uncommon and has been scarcely referred in the literature. We describe a family with two females affected by both NF1 and early-onset breast cancer, and a male with NF1. We evaluated whether the concomitance of both disorders could be attributed to a NF1 mutation and its supposed increased risk of breast cancer or to the concurrence of two NF1 and BRCA1/2 germline mutations. Mutation analyses identified a frameshift mutation in BRCA1 and a nonsense mutation in NF1. Our findings stress the importance of considering all phenotypic features in families with both NF1 and breast tumors. To offer a specific risk assessment and management of both conditions, NF1 and BRCA1/2 cancer predisposing genes should be analyzed.

Sex ratio distortion in offspring of families with BRCA1 or BRCA2 mutant alleles: an ascertainment bias phenomenon?

BACKGROUND: There has been controversy regarding whether BRCA1 germline mutations favor female births or whether the sex imbalances observed are attributable to ascertainment bias. Our aims were to compare the sex ratios among offspring of BRCA1-positive, BRCA2-positive, and BRCA-negative families undergoing genetic testing in clinical programs, and to determine whether ascertainment bias is responsible for the observed preponderance of female offspring. PATIENTS AND METHODS: A total of 145 breast and/or ovarian cancer families with mutations in BRCA1 (n = 83) or BRCA2 (n = 62), and 90 families without identifiable mutation were collected for the study from familial cancer clinics in Barcelona, Spain, and Boston, US. Sex ratio was analyzed among all births in the families and offspring of all (tested and obligate) carriers. In order to minimize the effect of family history of cancer, the analysis was also performed among offspring of the most recent generation of mutation-positive carriers who did not have affected children and compared with a control group comprised of the offspring of the most recent adult generation of non-carriers from families with a known mutation. RESULTS: There was a statistically higher proportion of female births in all groups (BRCA1 59% (95% CI = 57-61%), BRCA2 58% (56-61%), and BRCA-negative 59% (56-61%), respectively). The female preponderance persisted in analyses limited to offspring of BRCA1 and BRCA2 carriers (61% (57-65%), and 62% (58-66%), respectively), with no differences between the two mutation groups. In contrast, the excess of female offspring disappeared when ascertainment or recall biases were minimized, 44% (37-52%), and 39% (26-53%) for BRCA1; 51% (44-58%), and 46% (33-60%) for BRCA2. CONCLUSIONS: Our findings suggest that there is no asymmetry in birth outcomes among BRCA1 or BRCA2 mutations carriers. Rather ascertainment bias in families participating in genetic testing, or in the family history information they provide is likely to account for excess of female offspring previously reported.

[Haplotype of the BRCA2 6857delAA mutation in 4 families with breast/ovarian cancer]. / Análisis del haplotipo en portadores de la mutación 6857delAA en el gen BRCA2 en 4 familias con cáncer de mama u ovario hereditario.

BACKGROUND AND OBJECTIVE: It is estimated that 5-10% of all breast cancers are hereditary, mainly are due to germline mutations in the BRCA1 and BRCA2 genes. PATIENTS AND METHOD: A BRCA2 screening was carried out in familial breast/ovarian cancer at two centres in Spain and Chile. The 6857delAA mutation was identified in 3 Spanish families and one Chilean, all of them with Spanish ancestors. The BRCA2 haplotype of the 6857delAA carriers was analyzed using five microsatellite markers flanking the BRCA2 gene, spanning a region of 6 cM: cen-D13S260, D13S1698, (BRCA2), D13S171, D13S310 and D13S267-tel. RESULTS: Two families shared the allelic variants of the 5 microsatellites studied. Markers D13S260 and D13S267 differed in one allele in two families, respectively. The defined haplotype was absent in non-carriers from these families, and was not detected in 100 control chromosomes without the mutation. CONCLUSIONS: Our results suggest the existence of a common ancestry with the mutation originating in the Northeast of Spain. Given the migratory movements from Spain to Latin America, the screening of recurrent Spanish mutations can be useful in establishing a more rational and cost-effective analysis in such populations.

Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects.

We screened index cases from 410 Spanish breast/ovarian cancer families and 214 patients (19 of them males) with breast cancer for germ-line mutations in the BRCA1 and BRCA2 genes, using SSCP, PTT, CSGE, DGGE, and direct sequencing. We identified 60 mutations in BRCA1 and 53 in BRCA2. Of the 53 distinct mutations observed, 11 are novel and 12 have been reported only in Spanish families (41.5%). The prevalence of mutations in this set of families was 26.3%, but the percentage was higher in the families with breast and ovarian cancer (52.1%). The lowest proportion of mutations was found in the site-specific female breast cancer families (15.4%). Of the families with male breast cancer cases, 59.1% presented mutations in the BRCA2 gene. We found a higher frequency of ovarian cancer associated with mutations localized in the 5' end of the BRCA1 gene, but there was no association between the prevalence of this type of cancer and mutations situated in the ovarian cancer cluster region (OCCR) region of exon 11 of the BRCA2 gene. The mutations 187_188delAG, 330A>G, 5236G>A, 5242C>A, and 589_590del (numbered after GenBank U14680) account for 46.6% of BRCA1 detected mutations whereas 3036_3039del, 6857_6858del, 9254_9258del, and 9538_9539del (numbered after GenBank U43746) account for 56.6% of the BRCA2 mutations. The BRCA1 330A>G has a Galician origin (northwest Spain), and BRCA2 6857_6858del and 9254_9258del probably originated in Catalonia (northeast Spain). Knowledge of the spectrum of mutations and their geographical distribution in Spain will allow a more effective detection strategy in countries with large Spanish populations.

RNA analysis of eight BRCA1 and BRCA2 unclassified variants identified in breast/ovarian cancer families from Spain.

Germ-line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 account for a large proportion of hereditary breast/ovarian cancer families. A large number of disease-causing germ-line mutations and variants of unknown pathological significance have been identified in both genes. The majority of these variants have been studied only in genomic DNA and their effects at the mRNA level have not been reported. Our aim was to ascertain the pathological effect of six BRCA1 and two BRCA2 sequence unclassified variants by RNA analysis. Three of the BRCA1 variants are novel: IVS18+5G>A, IVS20-6_IVS20-4del and IVS22-2A>G. Three BRCA1 mutations showed aberrant splicing: Ala1693del, IVS18+5G>A and IVS22-2A>G. The variants G1706A, S1715N and IVS20-6_IVS20-4del in BRCA1, and T2515I and IVS25+9A>C in BRCA2 led to normal transcripts. We compared these RNA results with those obtained from two theoretical splicing prediction methods. The consensus values for the splice sequences (Shapiro and Senapathy 1987) involved in three of the BRCA1 splicing site variants agreed with the RNA results, lending support to the validity of this model. Moreover, we used previously established exonic splicing enhancer (ESE) sequences to ascertain whether the four exonic variants studied fell within predicted ESE motifs and whether they would disrupt ESE functions. Our results suggest that the splicing predictions based on this method are not definitive and should be considered with caution. This work highlights the importance of studying mutations at DNA and RNA levels in order to clarify their pathological effect. This information is essential for providing efficient counseling for breast/ovarian cancer families.

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain.

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.