SAT-295 An Extremely Rare Novel Missense Variant C.912G≫A; P.M304I in SOX3 Gene Is Responsible for X-Linked GH Deficiency in a Brazilian Boy Without Mental Retardation

SOX3 (SRY-related HMG-box gene 3), located in the X chromosome, spans only one exon and is expressed in the infundibulum, diencephalon and hypothalamus. Alterations in SOX3, mainly deletions or insertions in the polyalanine tract, were associated with mental retardation, isolated GH deficiency (IGHD) or combined pituitary hormone deficiencies (CPHD). Missense variants are rare and only two were reported. Our aim was to find a molecular cause in patients with pituitary hormone deficiency and determine genotype-phenotype correlation. Twenty-eight patients (15F:13M) 24 CPHD:4 IGHD were selected for the study. Whole blood DNA was extracted using the Salting Out method. Library preparation was performed following Agilent’s SureSelectXT customized gene panel protocol containing 654 genes known to cause endocrine diseases. Illumina NextSeq 500 platform was used for sequencing at SELA. Alignment to genome reference hg19 was performed using BWA-MEM. Variants were called with FreeBayes and annotated by Annovar. Allele frequency ≤1% for exonic regions was considered in 1000 Genomes, gnomAD, ABraOM and SELA populational databases for variant filtering. Family segregation was done using Sanger sequencing. RNA and protein analysis were performed using mfold and YASARA, respectively. Protein models were made by I-Tasser. SOX3 missense variant (c.912G>A/p.M304I) was found in one male patient, without mental retardation, diagnosed with IGHD at the age of 7 years. After GH replacement, he reached final height at the age of 18 within family target height. Pituitary image showed an ectopic posterior pituitary, hypoplastic anterior pituitary and thin pituitary stalk. SOX3 (c.912G>A/p.M304I) variant in hemizygous state was absent in populational data banks. In silico prediction algorithms SIFT, PolyPhen, and Mutation Assessor were predicted as damaging. Family segregation showed normal mother and sister carriers of the variant, while father, brother and uncle (from mother’s side), all phenotypically normal, did not harbor the variant. RNA In silico analysis pointed that the variant causes mRNA structure change. Protein stability dropped from 677.46 kcal/mol in wild type to 666.69 kcal/mol in p.M304I, making it less stable. Protein Interaction analysis with DNA binding motif (PDB 2LE4) required two times less energy in mutant (376.19 kcal/mol) than wild type protein (646.77 kcal/mol), leading to a less stable interaction. We conclude that one among 28 patients presented a rare novel variant in SOX3 associated to IGHD in a patient without mental retardation and compatible with an X-linked inheritance pattern.

Brugada Syndrome: Oligogenic or Mendelian Disease?

Brugada syndrome (BrS) is diagnosed by a coved-type ST-segment elevation in the right precordial leads on the electrocardiogram (ECG), and it is associated with an increased risk of sudden cardiac death (SCD) compared to the general population. Although BrS is considered a genetic disease, its molecular mechanism remains elusive in about 70–85% of clinically-confirmed cases. Variants occurring in at least 26 different genes have been previously considered causative, although the causative effect of all but the SCN5A gene has been recently challenged, due to the lack of systematic, evidence-based evaluations, such as a variant’s frequency among the general population, family segregation analyses, and functional studies. Also, variants within a particular gene can be associated with an array of different phenotypes, even within the same family, preventing a clear genotype–phenotype correlation. Moreover, an emerging concept is that a single mutation may not be enough to cause the BrS phenotype, due to the increasing number of common variants now thought to be clinically relevant. Thus, not only the complete list of genes causative of the BrS phenotype remains to be determined, but also the interplay between rare and common multiple variants. This is particularly true for some common polymorphisms whose roles have been recently re-evaluated by outstanding works, including considering for the first time ever a polygenic risk score derived from the heterozygous state for both common and rare variants. The more common a certain variant is, the less impact this variant might have on heart function. We are aware that further studies are warranted to validate a polygenic risk score, because there is no mutated gene that connects all, or even a majority, of BrS cases. For the same reason, it is currently impossible to create animal and cell line genetic models that represent all BrS cases, which would enable the expansion of studies of this syndrome. Thus, the best model at this point is the human patient population. Further studies should first aim to uncover genetic variants within individuals, as well as to collect family segregation data to identify potential genetic causes of BrS.

First confirmatory study on PTPRQ as an autosomal dominant non-syndromic hearing loss gene

Background Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*). Methods A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis. Results Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset. Conclusion Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss.

MARCAS DO PASSADO: MEMÓRIAS E SENTIMENTOS DE (EX) PORTADORES DE HANSENÍASE RESIDENTES EM UM ANTIGO “LEPROSÁRIO”

Resumo: Objetivo: descrever as principais memórias e sentimentos de “ex-portadores” de hanseníase residentes em um antigo “leprosário” localizado na região metropolitana do município de Belém, Estado do Pará. Metodologia: Trata-se de um estudo descritivo e exploratório, com abordagem qualitativa. Os dados foram coletados por meio de entrevistas em profundidade, no período de setembro a outubro de 2016, tendo como amostra de convivência onze (ex) portadores de hanseníase. A análise dos dados foi feita com base na análise de conteúdo de Bardin. Resultados: Emergiram três categorias: do medo à exclusão social: sentimentos diante descoberta da doença; o discurso do medo: isolamento compulsório e segregação familiar; Fantasmas do passado no presente: marcas sociais da doença. Considerações finais: As percepções acerca da doença evidenciam que marcas do passado ainda refletem no cotidiano social. As conotações negativas associadas à doença reforçam o preconceito e o estigma social, geram intensos abalos psíquicos e promovem constantes isolamentos sociais.Descritores: Hanseníase; Abrigo; Percepção; Estigma Social.THE MAIN MEMORIES AND FEELINGS OF EX –LEPROSY LIVING IN AN ANCIENT LEPROSARIUMObjective: Describe the main memories and feelings of ex –leprosy living in an ancient leprosarium located in the metropolitan region of Belém, State of Pará. Methodology: It is a descriptive and exploratory study with a qualitative approach. The data were collected through in-depth interviews, in the period from September to October of 2016, having as sample of coexistence, eleven ex-leprosy. The analysis of the data was made based content analysis of Bardin. Results: three categories emerged: From fear to social exclusion: feelings on the discovery of the disease; The discourse of fear: compulsory isolation and family segregation; Ghosts of the past in the present: social marks of disease. Conclusion: the perceptions about the disease show that marks of the past still reflect in social everyday life. The negative connotations associated with the disease reinforce prejudice and social stigma, generate intense psychic upsets and promote constant social isolation.Descriptors: Leprosy; Shelter; Perceptions; Social Stigma.MARCAS DEL PASADO: MEMORIAS Y SENTIMIENTOS DE (EX) PORTADORES DE LEPRA RESIDENTES EN UN ANTIGUO “LEPROSARIO”Objetivo: describir las principales memorias y sentimientos de “ex portadores” de lepra residentes en un antiguo “leprosario” ubicado en la región metropolitana del municipio de Belém, Estado de Pará. Metodología: Se trata de un estudio descriptivo y exploratorio, con enfoque cualitativo. Los datos fueron recolectados a través de entrevistas en profundidad, en el período de septiembre a octubre de 2016, con una muestra de convivencia once (ex) portadores de lepra. El análisis de los datos se basó en el análisis de contenido de Bardin. Resultados: Han surgido tres categorias: del miedo a la exclusión social: sentimientos ante descubrimiento de la enfermedad; El discurso del miedo: aislamiento obligatorio y segregación familiar; Fantasmas del pasado en el presente: marcas sociales de la enfermedad. Conclusión: las percepciones acerca de la enfermedad evidencian que las marcas del pasado todavía reflejan en el cotidiano social. Las connotaciones negativas asociadas a la enfermedad refuerzan el prejuicio y el estigma social, generan intensos sacudones psíquicos y promueven constantes aislamientos sociales.Descriptores: Lepra; Abrigo; Percepción; Estigma Social.

Chromosomal Location and Inheritance of Stem Rust Resistance Transferred from Hordeum bulbosum into Cultivated Barley (H. vulgare)

Stem rust, caused by Puccinia graminis f. sp. tritici, is an important disease on barley (Hordeum vulgare). Host resistance has effectively controlled stem rust, primarily through use of gene Rpg1. However, virulence to Rpg1 is present in North America, and a new race (TTKSK, or Ug99) from eastern Africa threatens barley production. A search for novel resistance was previously conducted, and an interspecific barley breeding line (212Y1) with introgressed chromatin from H. bulbosum was identified as carrying resistance to races MCCF and QCCJ. This study evaluated the inheritance of resistance in 212Y1 using populations from crosses to Morex (Rpg1 donor) and Q21861 (rpg4 donor) and the pathogen races MCCF (avirulent on Rpg1 and rpg4) and QCCJ (virulent on Rpg1 and avirulent on rpg4), and determined the chromosomal position of the introgression using genomic in situ hybridization (GISH) and chromosome-specific polymerase chain reaction (PCR)-based markers. Progeny from the 212Y1/Q21861 F2 population segregated for resistant and susceptible plants, indicating different gene loci. Genetic analyses of Morex/212Y1 F3 families fit a 7 homozygous resistant (HR):8 segregating:1 homozygous susceptible (HS) family segregation ratio to race MCCF, indicating that two genes controlled resistance. Plants in segregating families were in 3R:1S (Rpg1), 13R:3S (Rpg1+212Y1), and 1R:3S (212Y1) ratios. Genetic analyses of the same F3 families fit a 1HR:2 segregating:1HS family segregation ratio to race QCCJ, indicating monogenic inheritance. Plants in segregating families were in a 1R: 3S ratio, indicating recessive inheritance in 212Y1. The introgression from H. bulbosum into H. vulgare was positioned on chromosome 6HS based on GISH and the PCR-based markers. No known stem rust resistance gene has previously been mapped to that region. Thus, it is proposed to name this novel gene from H. bulbosum as rpg6.