Complete Loss Of Function Mutations Research Articles

The nanophthalmos protein TMEM98 inhibits MYRF self-cleavage and is required for eye size specification.

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.

Connective tissue alterations in Fkbp10-/- mice.

Osteogenesis imperfecta (OI) is an inherited brittle bone disorder characterized by bone fragility and low bone mass. Loss of function mutations in FK506-binding protein 10 (FKBP10), encoding the FKBP65 protein, result in recessive OI and Bruck syndrome, of which the latter is additionally characterized by joint contractures. FKBP65 is thought to act as a collagen chaperone, but it is unknown how loss of FKBP65 affects collagen synthesis and extracellular matrix formation. We evaluated the developmental and postnatal expression of Fkbp10 and analyzed the consequences of its generalized loss of function. Fkbp10 is expressed at low levels in E13.5 mouse embryos, particularly in skeletal tissues, and steadily increases through E17.5 with expression in not only skeletal tissues, but also in visceral tissues. Postnatally, expression is limited to developing bone and ligaments. In contrast to humans, with complete loss of function mutations, Fkbp10(-/-) mice do not survive birth, and embryos present with growth delay and tissue fragility. Type I calvarial collagen isolated from these mice showed reduced stable crosslink formation at telopeptide lysines. Furthermore, Fkbp10(-/-) mouse embryonic fibroblasts show retention of procollagen in the cell layer and associated dilated endoplasmic reticulum. These data suggest a requirement for FKBP65 function during embryonic connective tissue development in mice, but the restricted expression postnatally in bone, ligaments and tendons correlates with the bone fragility and contracture phenotype in humans.

Molecular defects of the GNRH receptor gene in Chinese patients with idiopathic hypogonadotropic hypogonadism and the severity of hypogonadism

To identify and determine the frequency of mutations in the coding region of the gonadotropin-releasing hormone receptor (GnRHR) gene in forty Chinese patients with normosmic idiopathic hypogonadotropic hypogonadism (IHH) and establish genotype/phenotype correlations where possible. The diagnosis of HH was based on absent or incomplete sexual development after 17 yr of age in girls and 18 yr in boys associated with low or normal levels of LH in both sexes and low levels of testosterone in males and of estradiol in females. All patients presented with a normal sense of smell in an olfactory specific test.40 IHH patients and 40 controls were screened for mutations in the coding sequence of GnRHR gene. The coding region of the GnRHR gene was amplified by PCR and directly sequenced. A missense mutation serine168arginine (S168R) located in the fourth transmembrane domain of the GnRH-R gene was identified in a homozygous state in one male with complete HH, the S168R mutation has been previously Shown to cause in the complete loss of receptor function because hormone binding to the receptor is completely impaired. In another patient, a compound heterozygous mutation (Gln106Arg and Arg262Gln) was identified in a male with partial HH, the Gln106Arg mutation located in the first extracellular loop of GnRH-R, this mutation decrease but not eliminate GnRH binding, while Arg262Gln mutation located in the third extracellular loop of GnRH-R and only decreases signal transduction. A good correlation between genotype and phenotype was found in our patients. The patient, who is homozygous for the completely inactivating S168R mutation, has complete HH. In addition, the affected patient who is compound heterozygotes for the Gln106Arg - Arg262Gln mutations, has partial HH. GnRHR mutations can be classified into partial or complete loss of function mutations. Partially inactivating substitutions of the GnRHR frequently found in familial hypogonadotrophic hypogonadism are Q106R and R262Q. comparison of compound heterozygous with homozygous patients suggests that their phenotype and the response to GnRH is determined by the GnRHR variant with the less severe loss of function.

Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) due to P450c21 (21-hydroxylase deficiency) is a common autosomal recessive disorder. This disorder is due to mutations in the CYP21A2 gene which is located at chromosome 6p21. The clinical features reflect the magnitude of the loss of function mutations. Individuals with complete loss of function mutations usually present in the neonatal period. The clinical features of individuals with mild loss of function mutations are predominantly due to androgen excess rather than adrenal insufficiency leading to an ascertainment bias favoring diagnosis in females. Treatment goals include normal linear growth velocity and “on-time” puberty in affected children. For adolescent and adult women, treatment goals include regularization of menses, prevention of progression of hirsutism, and fertility. This article will review key aspects regarding pathophysiology, diagnosis, and treatment of CAH.

Functional Characterization of Polymorphic Variants in the Human Apical Sodium‐dependent Bile Acid Transporter (ASBT; SLC10A2)

The apical sodium‐dependent bile acid transporter (ASBT) mediates the uptake of bile acids into intestinal enterocytes, supporting a critical role in normal bile acid homeostasis and maintenance of enterohepatic recirculation. Complete loss of function mutations in ASBT are associated with primary bile acid malaborption, characterized by diarrhea, steatorrhea, interruption of bile acid enterohepatic circulation, and reduced plasma cholesterol levels. We identified 6 nonsynonymous polymorphic variants within the exonic regions of the SLC10A2 gene from DNA derived from a large number of ethnically diverse healthy subjects. In general, variants tended to be infrequent and ethnic‐dependent; only one variant, 511GT (Ala171Ser), was found with a relatively common allele frequency (6.0%), in European‐Americans. A recombinant vaccinia‐based system was utilized to heterologously express wild‐type and variant ASBT variants in HeLa cells for functional studies using radiolabeled taurocholate as a substrate. Two variants, 292GA (Val98Ile) and 431GA (Cys144Tyr), were associated with significantly impaired taurocholate transport activity in vitro compared to wild‐type ASBT. Accordingly, polymorphisms in SLC10A2 may have important functional implications to normal bile acid homeostasis and enterohepatic recirculation. This work was supported in part by NIH grants GM54724 (RBK) and GM81363 (RHH).

UNC-97/PINCH is involved in the assembly of integrin cell adhesion complexes in Caenorhabditis elegans body wall muscle

UNC-97/PINCH is an evolutionarily conserved protein that contains five LIM domains and is located at cell–extracellular matrix attachment sites known as cell adhesion complexes. To understand the role of UNC-97/PINCH in cell adhesion, we undertook a combined genetic and cell biological approach to identify the steps required to assemble cell adhesion complexes in Caenorhabditis elegans. First, we have generated a complete loss of function mutation in the unc-97 coding region. unc-97 null mutants arrest development during embryogenesis and reveal that the myofilament lattice and its attachment structures, which include PAT-4/ILK (integrin-linked kinase) and integrin fail to assemble into properly organized arrays. Although in the absence of UNC-97/PINCH, PAT-4/ILK and integrin fail to organize normally, they are capable of colocalizing together at the muscle cell membrane. Alternatively, in integrin and pat-4 mutants, UNC-97/PINCH fails to localize to the muscle cell membrane and instead is found diffusely throughout the muscle cell cytoplasm. In agreement with mammalian studies, we show that LIM domain 1 of UNC-97/PINCH is required for its interaction with PAT-4/ILK in yeast two-hybrid assays. Additionally, we find, by LIM domain deletion analysis, that LIM1 is required for the localization of UNC-97/PINCH to cell adhesion complexes. Our results provide evidence that UNC-97/PINCH is required for the development of C. elegans and is required for the formation of integrin based adhesion structures.

Apaf1 in Chronic Myelogenous Leukemia (CML) Progression: Reduced Apaf1 Expression is Correlated with a H179R p53 Mutation During Clinical Blast Crisis

Chronic myelogenous leukemia (CML) is a stem cell disorder that eventually progresses to ablast crisis phase (BC) characterized by distorted apoptotic pathways. The exact mechanismleading to failure in apoptotic pathways during CML progression is unclear. In view of thecentral role of p53 and apaf1 in the apoptotic machinery we examined six human pairedchronic and BC phases samples for their expression.Real-time PCR (RQ-PCR) experiments showed an elevation of p53 mRNA in all patientsduring transition to BC. However, elevation of apaf1 during BC was observed in five patientsonly. In contrast, one patient displayed a significant 11.5-fold reduction of apaf1 expressionduring the transition to BC. No apaf1 promoter methylation was observed. The reduced apaf1expression was accompanied by a trans-dominant point mutation (H179R) in one p53 alleleand the loss of the other. This mutant p53, when tested using functionality assays, was unableto activate apaf1, consequently explaining the reduced expression observed in this patient.Furthermore, the same mutant failed to activate either genes involved in apoptotic or cellcycle arrest pathways, and can be considered as a complete loss of function mutation. Thisspecific mutation was reported in several types of cancer, but was not implicated in CML. Toconclude, in this study we have demonstrated mRNA elevation of p53 and apaf1 during CMLblast crisis, indicating that genes and proteins involved in cellular apoptosis might be involvedin disease progression/response to therapy. Moreover, the mutated p53 discovered in thepatient exhibiting lowered apaf1 expression provides, in a clinical case, the first correlationbetween p53 and apaf1 transcription regulation in humans.

ANDRE‐MAYER AWARD AW0002 The leptin‐melanocortin pathway in human physiology: insights from genetics

Considerable attention has been paid to the secular changes in food intake and physical activity that underlie the recent rise in the prevalence of obesity. However, there is compelling evidence that inter‐individual differences in susceptibility to obesity have strong genetic determinants. In the past decade, there have been major advances in our understanding of the molecular constituents of the pathways that control mammalian energy homeostasis. We and others have described six human obesity syndromes that arise from genetic defects. We have identified several patients with congenital leptin deficiency. These children are hyperphagic, develop severe disabling obesity, impaired T cell mediated immunity and hypogonadotropic hypogonadism. In a clinical trial of daily subcutaneous injections of recombinant human leptin, sustained, beneficial effects on appetite, fat mass, hyperinsulinaemia and hyperlipidaemia have been observed. Leptin administration also permits the full progression of appropriately timed puberty and reverses impaired T cell mediated immunity. We have recruited over 2000 severely obese children to the Genetics of Obesity Study (GOOS). Using a candidate gene approach, we have identified several loss of function mutations in the melanocortin 4 receptor (MC4R), which cause a dominantly inherited syndrome that accounts for up to 5% of patients with severe, early‐onset obesity, making this the commonest obesity syndrome to date. MC4R deficiency is characterized by hyperphagia, severe hyperinsulinaemia and increased linear growth and there is evidence for a genotype‐phenotype correlation, as complete loss of function mutations result in a more severe phenotype. The characterization of these syndromes and of patients with mutations in the genes encoding the leptin receptor, pro‐opiomelanocortin (POMC) and prohormone convertase ‐1, has provided a better mechanistic understanding of the regulation of appetite and body weight in humans, which will have implications for the treatment of obesity and other metabolic disorders.