Genotype-phenotype relationships in Berardinelli-Seip congenital lipodystrophy.

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A gene on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 70 affected subjects from 44 apparently unrelated pedigrees of diverse ethnic origin. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus, and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1 or BSCL2 and those with evidence against cosegregation with either chromosome 9 or 11 (designated BSCLX). BSCL2 appears to be a more severe disorder than BSCL1 with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment than those with BSCL1 or BSCLX (p<0.0001, OR 17.0, CI 3.6 to 79.0). The higher prevalence of intellectual impairment and the increased risk of premature death in BSCL2 compared to BSCL1 emphasise the importance of molecular diagnosis of this syndrome and have clear implications for genetic counselling.

Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.

Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), gamma3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.

Mutational analysis of activin/transforming growth factor-beta type I and type II receptor kinases in human pituitary tumors.

Genetic mutation or loss of activin/transforming growth factor-beta (TGFbeta) receptor function has been shown in human lymphoid, breast, and colorectal tumors as well as Hep2B and Mv1Lu cell lines. Although activin stimulates FSHbeta biosynthesis and secretion, a large percentage of human gonadotroph tumors have previously been demonstrated to be nonresponsive to characterized activin effects. This phenotype may be indicative of loss of functional cell surface receptors and/or intracellular signaling mediators of activin responses. Several studies examining the structure/function of type I and II receptors specific for ligands in the TGFbeta superfamily have delineated the critical regions for receptor intracellular kinase function. In the case of TGFbeta, inactivating mutations in these regions have been shown to render these receptors kinase deficient by a dominant negative phenotype and result in resistance to growth arrest. We therefore hypothesized that activin/TGFbeta cell surface receptors may act as tumor suppressors in human pituitary tumors, and that inactivating genetic mutations in the intracellular kinase region of this gene family may release pituicytes from normal growth suppression by activin through a similar mechanism. We used single stranded conformational polymorphism analysis to examine 2 intracellular regions required for type I receptor signaling by human Alk1-5 type I receptors as well as the entire coding region of 2 activin type II receptors and the TGFbeta type II receptor in 64 human pituitary tumors. A novel polymorphism was found in 45% of tumors at codon P117 of the ActRIIA gene and was used as a positive control for single stranded conformational polymorphism. One patient with a gonadotroph tumor had a confirmed A482V germline mutation in the Alk1 gene within kinase subdomains X-XI. No other mutations were detected in any tumor studied. These data suggest that somatic mutations within these intracellular kinase regions of type I/type II receptors are rare in human pituitary tumors.

Characteristics of growth hormone binding to liver microsomes of pregnant women.

Specific binding of hGH to liver microsomes of nonpregnant women and men is low, usually 10% of less in RRA. In pregnant women, however, we found that this binding is 10 times higher. The binding reaction shared the properties common to receptor systems: time, temperature and cation dependence, saturability and reversibility, hGH specific binding without cations was 10 times lower. The cross-reactions of hPRL and human placental lactogen with hGH were 0.49 +/- 0.16% and 0.10 +/- 0.05%, respectively. 125I-hPRL and 125I-human placental lactogen binding to microsomes of two controls and two pregnant women were very low and poorly reproducible. The Scatchard analysis revealed two hGH binding sites, one with an association constant (KA) of 2.7 +/- 0.1 x 10(10) M-1, and the other with a KA of 1.5 +/- 0.1 x 10(9) M-1. In one nonpregnant woman, we found a single hGH binding site with a KA of 1.5 x 10(9) M-1, confirming results previously reported in the literature. A hGH RRA was set up with microsomes of pregnant women. Acromegalic sera produced curves parallel to the hGH standard and pituitary dwarf serum had no 125I-hGH displacing activity. Sera of pregnant women produced curves divergent to the hGH standard and showed a 125I-hGH displacing activity 20 to 40 times higher than could be predicted by hGH levels determined by RIA. Cord sera and sera from puerperal women had similar hGH levels as determined by either RRA or RIA (r = 0.93, P less than 0.001, slope = 0.85, n = 25). Our results show the existence of specific GH receptors and serum factor(s) with high 125I-hGH displacing activity from these receptors in pregnancy. These findings must be related to several metabolic changes of pregnancy, such as glucose intolerance, hyperinsulinemia, increased lipolysis, and ketogenesis.

The "so-called" somatomedin B and growth hormone measured by radioimmunoassay in normal and cirrhotic individuals.

Serum somatomedin B (SmB) levels in cirrhotic individuals, 3.3 +/- 1.5 mg/l, were strikingly lower (P less than 0.001) than in normal subjects, 9.0 +/- 1.7 mg/l. SmB levels were clearly related to the levels of alpha 2-globulins in the cirrhotics (r = + 0.8, P less than 0.002). Serum SmB and growth hormone correlated negatively in a group of normal and cirrhotic individuals (r = -0.67, P less than 0.001). Direct measurements of serum SmB failed to reveal differences between hepatic, renal and peripheral veins. These findings suggest that: 1) SmB is produced by liver and/or normal liver function plays an important role in maintaining normal serum SmB levels; 2) SmB carrier proteins are reduced in the cirrhotics and 3) SmB is part of a negative feed-back system involving growth hormone.

Isolation and characterization of collagen-synthesizing polysomes from chick embryos.

Collagen-synthesizing polysomes were isolated by low-speed centrifugation of the post-mitochondrial supernatant of chick homogenates. Electron microscopy of the fraction thus isolated shows it to be exclusively composed of ribosomes. Amino acid incorporation in vitro showed that these particles were efficient in the incorporation of proline, but not tryptophan, as opposed to ribosomes obtained from the supernatant of the low-speed centrifugation. The incorporation process was highly dependent on GTP, and exibited an optimal Mg2+concentration of 5.6mM. The reaction was inhibited by RNase, elongation inhibitors as anysomycin, sparsomycin, fusidic acid and GDPCP. It was also moderately inhibited by initiation inhibitors such as aurintricarboxilic acid and pyrocatechol violet. The product of the incorporation was characterized as collagen by its sensitivity towards purified collagenase, lack of tryptophan, chromatography in CM-cellulose and molecular sieve chromatography in Sephadex G-200.