The hospital Israelita Albert Einstein standards for constitutional sequence variants classification: version 2023.

BACKGROUND: Next-generation sequencing has had a significant impact on genetic disease diagnosis, but the interpretation of the vast amount of genomic data it generates can be challenging. To address this, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology have established guidelines for standardized variant interpretation. In this manuscript, we present the updated Hospital Israelita Albert Einstein Standards for Constitutional Sequence Variants Classification, incorporating modifications from leading genetics societies and the ClinGen initiative. RESULTS: First, we standardized the scientific publications, documents, and other reliable sources for this document to ensure an evidence-based approach. Next, we defined the databases that would provide variant information for the classification process, established the terminology for molecular findings, set standards for disease-gene associations, and determined the nomenclature for classification criteria. Subsequently, we defined the general rules for variant classification and the Bayesian statistical reasoning principles to enhance this process. We also defined bioinformatics standards for automated classification. Our workgroup adhered to gene-specific rules and workflows curated by the ClinGen Variant Curation Expert Panels whenever available. Additionally, a distinct set of specifications for criteria modulation was created for cancer genes, recognizing their unique characteristics. CONCLUSIONS: The development of an internal consensus and standards for constitutional sequence variant classification, specifically adapted to the Brazilian population, further contributes to the continuous refinement of variant classification practices. The aim of these efforts from the workgroup is to enhance the reliability and uniformity of variant classification.

Brain monoamine vesicular transport disease caused by homozygous SLC18A2 variants: A study in 42 affected individuals.

PURPOSE: Brain monoamine vesicular transport disease is an infantile-onset movement disorder that mimics cerebral palsy. In 2013, the homozygous SLC18A2 variant, p.Pro387Leu, was first reported as a cause of this rare disorder, and dopamine agonists were efficient for treating affected individuals from a single large family. To date, only 6 variants have been reported. In this study, we evaluated genotype-phenotype correlations in individuals with biallelic SLC18A2 variants. METHODS: A total of 42 affected individuals with homozygous SLC18A2 variant alleles were identified. We evaluated genotype-phenotype correlations and the missense variants in the affected individuals based on the structural modeling of rat VMAT2 encoded by Slc18a2, with cytoplasm- and lumen-facing conformations. A Caenorhabditis elegans model was created for functional studies. RESULTS: A total of 19 homozygous SLC18A2 variants, including 3 recurrent variants, were identified using exome sequencing. The affected individuals typically showed global developmental delay, hypotonia, dystonia, oculogyric crisis, and autonomic nervous system involvement (temperature dysregulation/sweating, hypersalivation, and gastrointestinal dysmotility). Among the 58 affected individuals described to date, 16 (28%) died before the age of 13 years. Of the 17 patients with p.Pro237His, 9 died, whereas all 14 patients with p.Pro387Leu survived. Although a dopamine agonist mildly improved the disease symptoms in 18 of 21 patients (86%), some affected individuals with p.Ile43Phe and p.Pro387Leu showed milder phenotypes and presented prolonged survival even without treatment. The C. elegans model showed behavioral abnormalities. CONCLUSION: These data expand the phenotypic and genotypic spectra of SLC18A2-related disorders.

Congenital limb deficiency: Genetic investigation of 44 individuals presenting mainly longitudinal defects in isolated or syndromic forms.

Congenital limb deficiency (CLD), one of the most common congenital anomalies, is characterized by hypoplasia/aplasia of one or more limb bones and can be isolated or syndromic. The etiology in CLD is heterogeneous, including environmental and genetic factors. A fraction remains with no etiological factor identified. We report the study of 44 Brazilian individuals presenting isolated or syndromic CLD, mainly with longitudinal defects. Genetic investigation included particularly next-generation sequencing (NGS) and/or chromosomal microarray. The overall diagnostic yield was 45.7%, ranging from 60.9% in the syndromic to 16.7% in the non-syndromic group. In TAR syndrome, a common variant in 3´UTR of RBM8A, in trans with 1q21.1 microdeletion, was detected, corroborating the importance of this recently reported variant in individuals of African ancestry. NGS established a diagnosis in three individuals in syndromes recently reported or still under delineation (an acrofacial dysostosis, Coats plus and Verheij syndromes), suggesting a broader phenotypic spectrum in these disorders. Although a low rate of molecular detection in non-syndromic forms was observed, it is still possible that variants in non-coding regions and small CNVs, not detected by the techniques applied in this study, could play a role in the etiology of CLD.

Phenotype-genotype analysis of 242 individuals with RASopathies: 18-year experience of a tertiary center in Brazil.

We report the clinical and molecular data of a large cohort comprising 242 individuals with RASopathies, from a single Tertiary Center in Brazil, the largest study from Latin America. Noonan syndrome represented 76% of the subjects, with heterozygous variants in nine different genes, mainly PTPN11, SOS1, RAF1, LZTR1, and RIT1, detected by Sanger and next-generation sequencing. The latter was applied to 126 individuals, with a positive yield of 63% in genes of the RAS/MAPK cascade. We present evidence that there are some allelic differences in PTPN11 across distinct populations. We highlight the clinical aspects that pose more medical concerns, such as the cardiac anomalies, bleeding diathesis and proliferative lesions. The genotype-phenotype analysis between the RASopathies showed statistically significant differences in some cardinal features, such as craniofacial and cardiac anomalies, the latter also statistically significant for different genes in Noonan syndrome. We present two individuals with a Noonan syndrome phenotype, one with an atypical, structural cardiac defect, harboring variants in genes mainly associated with isolated hypertrophic cardiomyopathy and discuss the role of these variants in their phenotype.

Hemorrhagic stroke and renovascular hypertension with Grange syndrome arising from a novel pathogenic variant in YY1AP1.

Pediatric hypertension can cause hypertensive emergencies, including hemorrhagic stroke, contributing to rare but serious childhood morbidity and mortality. Renovascular hypertension (RVH) is one of the major causes of secondary hypertension in children. Grange syndrome (MIM#602531) is a rare disease characterized by multiple stenosis or occlusion of the renal, abdominal, coronary, and cerebral arteries, which can cause phenotypes of RVH and fibromuscular dysplasia (MIM#135580). We report the case of a 7-year-old girl with Grange syndrome who showed RVH and multiple seizure episodes. At 1 year of age, she experienced seizures and sequential hemiparesis caused by a left thalamic hemorrhage without cerebral vascular anomalies. Chronic hypertension was observed, and abdominal computed tomography angiography showed characteristic bilateral renal artery stenosis. Whole-exome sequencing revealed a novel homozygous pathogenic variant in the YY1AP1 gene (NM_001198903.1: c.1169del: p.Lys390Argfs*12). Biallelic YY1AP1 mutations are known to cause Grange syndrome. Unlike previously reported patients, our patient presented with intracerebral hemorrhagic stroke without anomalous brain artery or bone fragility. The phenotype in our patient may help better understand this ultra-rare syndrome. Grange syndrome should be considered in patients presenting with childhood-onset hypertension and/or hemorrhagic stroke for early clinical intervention.

Clinical and radiological findings in Brazilian patients with mucolipidosis types II/III.

OBJECTIVE: The present study aims to provide orientation for clinicians and radiologists to recognize the most prevalent findings leading to diagnosis in mucolipidosis from a description of the natural history of five Brazilian cases. MATERIALS AND METHODS: We conducted an observational and retrospective study of five patients with clinical and radiological diagnosis of mucolipidosis. Clinical evaluation consisted of information obtained from records and including physical, neurologic, and dysmorphic evaluations. Radiologic studies consisted of complete skeletal radiographs of all patients. Enzyme assessment was performed for confirmation of the diagnosis. RESULTS: The five patients were referred for genetic evaluation due to disproportionate short stature with short trunk accompanied by waddling gait. Age at referral varied from 11 months to 28 years. The most prevalent findings were joint restriction (4/5 patients), neuropsychomotor developmental delay (3/5), coarse facies (2/5), hypertrophic cardiomyopathy (2/5), and mental retardation (1/4 patients). The most common radiological findings were anterior beaking of the vertebral bodies (5/5), shallow acetabular fossae (5/5), epiphyseal dysplasia (5/5), platyspondyly (4/5), pelvic dysplasia (4/5), decreased bone mineralization (4/5), scoliosis (3/5), wide and oar-shaped ribs (3/5), generalized epiphyseal ossification delay (3/5), and hypoplasia of basilar portions of ilea (3/5). Enzyme assessment showed α-iduronidase, α-mannosidase, ß-glucuronidase, hexosaminidase A, and total hexosaminidase increased in plasma and normal glycosaminoglycans concentration. One patient was clinically classified as ML II and four patients as ML III. CONCLUSIONS: The follow-up of five patients showed the typical clinical and radiological findings allowing the diagnosis, thus improving clinical management and providing adequate genetic counseling. Clinicians and radiologists can take advantage of the information from this work, enhancing their differential diagnosis ability.

Parental segregation study reveals rare benign and likely benign variants in a Brazilian cohort of rare diseases.

Genomic studies may generate massive amounts of data, bringing interpretation challenges. Efforts for the differentiation of benign and pathogenic variants gain importance. In this article, we used segregation analysis and other molecular data to reclassify to benign or likely benign several rare clinically curated variants of autosomal dominant inheritance from a cohort of 500 Brazilian patients with rare diseases. This study included only symptomatic patients who had undergone molecular investigation with exome sequencing for suspected diseases of genetic etiology. Variants clinically suspected as the causative etiology and harbored by genes associated with highly-penetrant conditions of autosomal dominant inheritance underwent Sanger confirmation in the proband and inheritance pattern determination because a "de novo" event was expected. Among all 327 variants studied, 321 variants were inherited from asymptomatic parents. Considering segregation analysis, we have reclassified 51 rare variants as benign and 211 as likely benign. In our study, the inheritance of a highly penetrant variant expected to be de novo for pathogenicity assumption was considered as a non-segregation and, therefore, a key step for benign or likely benign classification. Studies like ours may help to identify rare benign variants and improve the correct interpretation of genetic findings.

Genomic study of nonsyndromic hearing loss in unaffected individuals: Frequency of pathogenic and likely pathogenic variants in a Brazilian cohort of 2,097 genomes.

Hearing loss (HL) is a common sensory deficit in humans and represents an important clinical and social burden. We studied whole-genome sequencing data of a cohort of 2,097 individuals from the Brazilian Rare Genomes Project who were unaffected by hearing loss to investigate pathogenic and likely pathogenic variants associated with nonsyndromic hearing loss (NSHL). We found relevant frequencies of individuals harboring these alterations: 222 heterozygotes (10.59%) for sequence variants, 54 heterozygotes (2.58%) for copy-number variants (CNV), and four homozygotes (0.19%) for sequence variants. The top five most frequent genes and their corresponding combined allelic frequencies (AF) were GJB2 (AF = 1.57%), STRC (AF = 1%), OTOA (AF = 0.69%), TMPRSS3 (AF = 0.41%), and OTOF (AF = 0.29%). The most frequent sequence variant was GJB2:c.35del (AF = 0.72%), followed by OTOA:p. (Glu787Ter) (AF = 0.61%), while the most recurrent CNV was a microdeletion of 57.9 kb involving the STRC gene (AF = 0.91%). An important fraction of these individuals (n = 104; 4.96%) presented variants associated with autosomal dominant forms of NSHL, which may imply the development of some hearing impairment in the future. Using data from the heterozygous individuals for recessive forms and the Hardy-Weinberg equation, we estimated the population frequency of affected individuals with autosomal recessive NSHL to be 1:2,222. Considering that the overall prevalence of HL in adults ranges from 4-15% worldwide, our data indicate that an important fraction of this condition may be associated with a monogenic origin and dominant inheritance.

Whole-genome sequencing of 1,171 elderly admixed individuals from São Paulo, Brazil.

As whole-genome sequencing (WGS) becomes the gold standard tool for studying population genomics and medical applications, data on diverse non-European and admixed individuals are still scarce. Here, we present a high-coverage WGS dataset of 1,171 highly admixed elderly Brazilians from a census-based cohort, providing over 76 million variants, of which ~2 million are absent from large public databases. WGS enables identification of ~2,000 previously undescribed mobile element insertions without previous description, nearly 5 Mb of genomic segments absent from the human genome reference, and over 140 alleles from HLA genes absent from public resources. We reclassify and curate pathogenicity assertions for nearly four hundred variants in genes associated with dominantly-inherited Mendelian disorders and calculate the incidence for selected recessive disorders, demonstrating the clinical usefulness of the present study. Finally, we observe that whole-genome and HLA imputation could be significantly improved compared to available datasets since rare variation represents the largest proportion of input from WGS. These results demonstrate that even smaller sample sizes of underrepresented populations bring relevant data for genomic studies, especially when exploring analyses allowed only by WGS.

Reproductive alternatives for patients with dystrophic epidermolysis bullosa.

Epidermolysis bullosa describes a group of skin conditions caused by mutations in genes encoding proteins related to dermal-epidermal adhesion. In the United States, 50 cases of epidermolysis bullosa per 1 million live births are estimated, 92% of which classified as simplex, 5% dystrophic, 1% junctional and 2% non-classified. Dystrophic epidermolysis bullosa is associated with autosomal, dominant and recessive inheritance. Epidermolysis bullosa causes severe psychological, economic and social impacts, and there is currently no curative therapy, only symptom control. Embryonic selection is available for epidermolysis bullosa patients in order to prevent perpetuation of the condition in their offspring.

Natural history of 39 patients with Achondroplasia.

OBJECTIVES: To characterize the natural history of 39 achondroplastic patients diagnosed by clinical, radiological and molecular assessments. METHODS: Observational and retrospective study of 39 patients who were attended at a public tertiary level hospital between 1995 and 2016. RESULTS: Diagnosis was made prenatally in 11 patients, at birth in 9 patients and within the first year of life in 13 patients. The most prevalent clinical findings were short stature, high forehead, trident hands, genu varum and macrocephaly. The most prevalent radiographic findings were rhizomelic shortening of the long bones and narrowing of the interpediculate distance of the caudal spine. There was motor developmental delay in 18 patients and speech delay in 16 patients. The most common clinical intercurrences were middle ear dysfunction, sleep apnea, limb pain and obesity from 2 to 9 years of age. One patient was large for the gestational age but did not develop obesity. One patient developed hydrocephalus at 10 years old. The current age of the patients varies from 15 months to 36 years. The molecular study performed by Sanger sequencing of the common heterozygous mutation 1138G>A in FGFR3 was positive in all patients. Four cases were inherited, and 35 were sporadic (paternal age from 19 to 66 years). CONCLUSIONS: The diagnoses were made early based on clinical and radiographic findings. All cases were confirmed molecularly. Despite presenting a benign course, it is necessary to establish a systematic protocol for the surveillance of these patients due to the common clinical intercurrences.

Reproductive alternatives for patients with dystrophic epidermolysis bullosa / Opções reprodutivas para pacientes com epidermólise bolhosa distrófica

ABSTRACT Epidermolysis bullosa describes a group of skin conditions caused by mutations in genes encoding proteins related to dermal-epidermal adhesion. In the United States, 50 cases of epidermolysis bullosa per 1 million live births are estimated, 92% of which classified as simplex, 5% dystrophic, 1% junctional and 2% non-classified. Dystrophic epidermolysis bullosa is associated with autosomal, dominant and recessive inheritance. Epidermolysis bullosa causes severe psychological, economic and social impacts, and there is currently no curative therapy, only symptom control. Embryonic selection is available for epidermolysis bullosa patients in order to prevent perpetuation of the condition in their offspring.

RESUMO O termo "epidermólise bolhosa" descreve um grupo de afecções cutâneas causadas por mutações em genes que codificam proteínas relacionadas à aderência dermoepidérmica. Nos Estados Unidos, estima-se a ocorrência de 50 casos de epidermólise bolhosa por 1 milhão de nascidos vivos, sendo 92% deles da forma simples, 5% da forma distrófica, 1% da forma juncional e 2% não classificados. A epidermólise bolhosa do tipo distrófica foi associada a padrões autossômicos, dominante e recessivo. A epidermólise bolhosa causa sérios impactos psicológicos, econômicos e sociais, e não há tratamento curativo atualmente − apenas controle dos sintomas. A seleção embrionária é disponível para portadores de epidermólise bolhosa, a fim de evitar a perpetuação da condição em seus descendentes.

Natural history of 39 patients with Achondroplasia

OBJECTIVES: To characterize the natural history of 39 achondroplastic patients diagnosed by clinical, radiological and molecular assessments. METHODS: Observational and retrospective study of 39 patients who were attended at a public tertiary level hospital between 1995 and 2016. RESULTS: Diagnosis was made prenatally in 11 patients, at birth in 9 patients and within the first year of life in 13 patients. The most prevalent clinical findings were short stature, high forehead, trident hands, genu varum and macrocephaly. The most prevalent radiographic findings were rhizomelic shortening of the long bones and narrowing of the interpediculate distance of the caudal spine. There was motor developmental delay in 18 patients and speech delay in 16 patients. The most common clinical intercurrences were middle ear dysfunction, sleep apnea, limb pain and obesity from 2 to 9 years of age. One patient was large for the gestational age but did not develop obesity. One patient developed hydrocephalus at 10 years old. The current age of the patients varies from 15 months to 36 years. The molecular study performed by Sanger sequencing of the common heterozygous mutation 1138G>A in FGFR3 was positive in all patients. Four cases were inherited, and 35 were sporadic (paternal age from 19 to 66 years). CONCLUSIONS: The diagnoses were made early based on clinical and radiographic findings. All cases were confirmed molecularly. Despite presenting a benign course, it is necessary to establish a systematic protocol for the surveillance of these patients due to the common clinical intercurrences.