Short stature caused by SHOX gene haploinsufficiency: from diagnosis to treatment

Estudos realizados em pacientes portadores de deleções parciais dos cromossomos sexuais permitiram a caracterização do SHOX, gene localizado na região pseudoautossômica no braço curto dos cromossomos sexuais, fundamental na determinação da altura normal. A perda de uma cópia deste gene na síndrome de Turner (ST) explica dois terços da baixa estatura observada nesta síndrome. A haploinsuficiência do SHOX é detectada em 77% dos pacientes com discondrosteose de Leri-Weill, uma forma comum de displasia esquelética de herança autossômica dominante e em 3% das crianças com baixa estatura idiopática (BEI), tornando os defeitos neste gene a principal causa monogênica de baixa estatura. A medida da altura sentada em relação à altura total (Z da AS/AT para idade e sexo) é uma forma simples de identificar a desproporção corpórea e, associada ao exame cuidadoso do paciente e de outros membros da família, auxilia na seleção de pacientes para o estudo molecular do SHOX. O uso de hormônio de crescimento (GH) está bem estabelecido na ST e em razão da causa comum da baixa estatura com o de crianças com defeitos isolados do SHOX o tratamento destes pacientes com GH é também proposto. Neste artigo será revisado os aspectos clínicos, moleculares e terapêuticos da haploinsuficiência do SHOX.Studies involving patients with short stature and partial deletion of sex chromosomes identified SHOX gene in the pseudoautosomal region of the X and Y chromosomes. SHOX haploinsufficiency is an important cause of short stature in a diversity of clinical conditions. It explains 2/3 of short stature observed in Turner syndrome (TS) patients. Heterozygous mutations in SHOX are observed in 77% of patients with Leri-Weill dyschondrosteosis, a common dominant inherited skeletal dysplasia and in 3% of children with idiopathic short stature, indicating that SHOX defects are the most frequent monogenetic cause of short stature. The sitting height/height ratio (SH/H) standard deviation score is a simple way to assess body proportions and together with a careful exam of other family members, effectively selected a group of patients that presented a high frequency of SHOX mutations. Growth hormone treatment of short stature due to TS is well established and considering the common etiology of short stature in patients with isolated defects of SHOX gene, this treatment is also proposed for these patients. Here, we review clinical, molecular and therapeutic aspects of SHOX haploinsufficiency.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Evaluation of SHOX defects in the era of next‐generation sequencing

Short stature homeobox (SHOX) haploinsufficiency is a frequent cause of short stature. Despite advances in sequencing technologies, the identification of SHOX mutations continues to be performed using standard methods, including multiplex ligation‐dependent probe amplification (MLPA) followed by Sanger sequencing. We designed a targeted panel of genes associated with growth impairment, including SHOX genomic and enhancer regions, to improve the resolution of next‐generation sequencing for SHOX analysis. We used two software packages, CONTRA and Nexus Copy Number, in addition to visual analysis to investigate the presence of copy number variants (CNVs). We evaluated 15 patients with previously known SHOX defects, including point mutations, deletions and a duplication, and 77 patients with idiopathic short stature (ISS). The panel was able to confirm all known defects in the validation analysis. During the prospective evaluation, we identified two new partial SHOX deletions (one detected only by visual analysis), including an intragenic deletion not detected by MLPA. Additionally, we were able to determine the breakpoints in four cases. Our results show that the designed panel can be used for the molecular investigation of patients with ISS, and it may even detect CNVs in SHOX and its enhancers, which may be present in a significant fraction of patients.Copy number variants analyses and Sanger sequencing of breakpoint regions in Case 11, which has a heterozygous deletions involving exons 4, 5, and 6a of short stature homeobox (SHOX).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151254/1/cge13587.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151254/2/CGE_13587-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151254/3/cge13587_am.pd

Targeted massively parallel sequencing panel to diagnose genetic endocrine disorders in a tertiary hospital

Objectives: To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods: Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results: Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion: The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling

Long‐term outcomes and molecular analysis of a large cohort of patients with 46,XY disorder of sex development due to partial gonadal dysgenesis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145419/1/cen13717_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145419/2/cen13717.pd

Post-receptor IGF1 insensitivity restricted to the MAPK pathway in a Silver-Russell syndrome patient with hypomethylation at the imprinting control region on chromosome 11

Background: Hypomethylation of the paternal imprinting center region 1 (ICR1) is the most frequent molecular cause of Silver-Russell syndrome (SRS). Clinical evidence suggests that patients with this epimutation have mild IGF1 insensitivity. Objective: To assess in vitro IGF1 action in fibroblast culture from a patient with SRS and IGF1 insensitivity. Methods: Fibroblast cultures from one patient with SRS due to ICR1 demethylation and controls were established. The SRS patient has severe growth failure, elevated IGF1 level, and poor growth rate during human recombinant GH treatment. IGF1 action was assessed by cell proliferation, AKT, and p42/44-MAPK phosphorylation. Gene expression was determined by real-time PCR. Results: Despite normal IGF1R sequence and expression, fibroblast proliferation induced by IGF1 was 50% lower in SRS fibroblasts in comparison with controls. IGF1 and insulin promoted a p42/44-MAPK activation in SRS fibroblasts 40 and 36%, respectively, lower than that in control fibroblasts. On the other hand, p42/44-MAPK activation induced by EGF stimulation was only slightly reduced (75% in SRS fibroblasts in comparison with control), suggesting a general impairment in MAPK pathway with a greater impairment of the stimulation induced by insulin and IGF1 than by EGF. A PCR array analysis disclosed a defect in MAPK pathway characterized by an increase in DUSP4 and MEF2C gene expressions in patient fibroblasts. Conclusion: A post-receptor IGF1 insensitivity was characterized in one patient with SRS and ICR1 hypomethylation. Although based on one unique severely affected patient, these results raise an intriguing mechanism to explain the postnatal growth impairment observed in SRS patients that needs confirmation in larger cohorts.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [08/57915-2, 05/04726-0, 05/50144-2]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [142062/06-5, 301339/2008-9, 300938/06-3, 307951/06-5

Homozygous Inactivating Mutation In Nanos3 In Two Sisters With Primary Ovarian Insufficiency.

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.201478746

A 46,XX testicular disorder of sex development caused by a Wilms’ tumour Factorâ 1 (WT1) pathogenic variant

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146930/1/cge13459_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146930/2/cge13459.pd

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca2 + pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition