Felix Schreiner

MKRN3 Mutations in Familial Central Precocious Puberty

Loss-of-function mutations in the gene encoding the makorin RING finger protein 3 (MKRN3) have recently been reported to underlie familial cases of central precocious puberty (CPP). The imprinted MKRN3 gene is expressed only from the paternal allele, and mutations inherited from the father affect boys and girls equally, which is in contrast to the known female preponderance in idiopathic CPP. By screening a series of 6 families and 1 male patient with idiopathic CPP, we identified 2 further families carrying loss-of-function mutations in MKRN3, the previously reported variant c.475_476insC (p.Ala162Glyfs*14) and a novel one, c.331G>T (p.Glu111*). We conclude that MKRN3 mutations appear to be a frequent cause of familial CPP and, considering the imprinted mode of inheritance, may also account for a certain proportion of isolated CPP cases. Remarkably, four out of six MKRN3 mutations described so far encode either a stop codon or a frameshift followed by a premature stop codon. Consequently, there may be less severe mutations that possibly associate with more subtle phenotypes, which could even explain variation within the physiological range. Mutation screening in larger cohorts is necessary in order to estimate the real prevalence of MKRN3 mutations in idiopathic CPP.

Methylation of L1Hs promoters is lower on the inactive X, has a tendency of being higher on autosomes in smaller genomes and shows inter-individual variability at some loci

LINE-1 repeats account for ∼17% of the human genome. Little is known about their individual methylation patterns, because their repetitive, almost identical sequences make them difficult to be individually targeted. Here, we used bisulfite conversion to study methylation at individual LINE-1 repeats. The loci studied included 39 X-linked loci and 5 autosomal loci. On the X chromosome in women, we found statistically significant less methylation at almost all L1Hs compared with men. Methylation at L1P and L1M did not correlate with the inactivation status of the host DNA, while the majority of L1Hs that were possible to be studied lie in inactivated regions. To investigate whether the male–female differences at L1Hs on the X are linked to the inactivation process itself rather than to a mere influence of gender, we analyzed six of the L1Hs loci on the X chromosome in Turners and Klinefelters which have female and male phenotype, respectively, but with reversed number of X chromosomes. We could confirm that all samples with two X chromosomes are hypomethylated at the L1Hs loci. Therefore, the inactive X is hypomethylated at L1Hs; the latter could play an exclusive role in the X chromosome inactivation process. At autosomal L1Hs, methylation levels showed a correlation tendency between methylation level and genome size, with higher methylation observed at most loci in individuals with one X chromosome and the lowest in XXY individuals. In summary, loci-specific LINE-1 methylation levels show considerable plasticity and depend on genomic position and constitution.

False negative 17-hydroxyprogesterone screening in children with classical congenital adrenal hyperplasia

We report 5 out of 214 children with classical congenital adrenal hyperplasia (CAH) that was not detected by neonatal 17-Hydroxyprogesterone screening. Therefore, diagnosis was only based on a suspect clinical picture and subsequent re-evaluation. In addition to 3 patients suffering from the simple virilizing form of CAH and not reported so far, the remaining 2 children whose CAH was missed by the screening suffered from the severe salt-wasting form. This report underlines the importance of a careful clinical investigation of newborns to detect signs of genital virilization. The differential diagnosis of classical CAH should be kept in mind even if neonatal screening is reported to be normal.

Infancy-Onset T1DM, Short Stature, and Severe Immunodysregulation in Two Siblings With a Homozygous LRBA Mutation

CONTEXT Type 1 diabetes mellitus (T1DM) is caused by autoimmunity against pancreatic β-cells. Although a significant number of T1DM patients have or will develop further autoimmune disorders during their lifetime, coexisting severe immunodysregulation is rare. OBJECTIVE Presuming autosomal-recessive inheritance in a complex immunodysregulation disorder including T1DM and short stature in two siblings, we performed whole-exome sequencing. CASE PRESENTATION Two Libyan siblings born to consanguineous parents were presented to our diabetology department at ages 12 and 5 years, respectively. Apart from T1DM diagnosed at age 2 years, patient 1 suffered from chronic restrictive lung disease, mild enteropathy, hypogammaglobulinemia, and GH deficiency. Fluorescence-activated cell sorting analysis revealed B-cell deficiency. In addition, CD4(+)/CD25(+) and CD25(high)/FoxP3(+) cells were diminished, whereas an unusual CD25(-)/FoxP3(+) population was detectable. The younger brother, patient 2, also developed T1DM during infancy. Although his enteropathy was more severe and electrolyte derangements repeatedly led to hospitalization, he did not have significant pulmonary problems. IgG levels and B-lymphocytes were within normal ranges. RESULTS By whole-exome sequencing we identified a homozygous truncating mutation (c.2445_2447del(C)3ins(C)2, p.P816Lfs*4) in the lipopolysaccharide-responsive beige-like anchor (LRBA) gene in both siblings. The diagnosis of LRBA deficiency was confirmed by a fluorescence-activated cell sorting-based immunoassay showing the absence of LRBA protein in phytohemagglutinin-stimulated peripheral blood mononuclear cells. CONCLUSION We identified a novel truncating LRBA mutation in two siblings with T1DM, short stature, and severe immunodysregulation. LRBA mutations have previously been reported to cause multiorgan autoimmunity and immunodysfunction. In light of the variable phenotypes reported so far in LRBA-mutant individuals, LRBA deficiency should be considered in all patients presenting with T1DM and signs of severe immunodysregulation.

DNA methylation signature in peripheral blood reveals distinct characteristics of human X chromosome numerical aberrations

BackgroundAbnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infinium assay on individuals with four karyotypes: 45,X, 46,XY, 46,XX, and 47,XXY.ResultsDNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci. Differentially methylated CpGs, in most cases, have intermediate methylation levels and tend to occur outside CpG islands, especially in individuals with Turner syndrome. The X inactivation process appears to be less effective in Klinefelter syndrome as methylation on the X was decreased compared to normal female samples. In a large number of individuals, we verified several loci by pyrosequencing and observed only weak inter-loci correlations between the verified regions. This suggests a certain stochastic/random contribution to the methylation changes at each locus. Interestingly, methylation patterns on some PAR2 loci differ between male and Turner syndrome individuals and between female and Klinefelter syndrome individuals, which possibly contributed to this distinguished and unique autosomal methylation patterns in Turner and Klinefelter syndrome individuals.ConclusionsThe presented data clearly show that gain or loss of an X chromosome results in different epigenetic effects, which are not necessary opposite.

Insulin-Like Growth Factor-I in Cord Blood Is Predictive of Catch-Up Growth in Monozygotic Twins with Discordant Growth

OBJECTIVE The aim was to investigate the growth of monozygotic twins with discordant birth weights and the predictive value of birth-weight, birth-length, and cord-blood concentration of growth factors on their catch-up growth up to the age of 4 yr. PATIENTS AND METHODS Twenty-five monozygotic twin-pairs [14 with an intertwin birth-weight sd score (SDS) difference>1] were studied at birth and at 4 yr of age. In all pairs, several parameters including IGF-I were analyzed in cord blood, and in 20 pairs parameters were analyzed again at 4 yr of age. Intertwin differences (Δ) in birth weight, birth length, and growth at 3, 6, 12, 24, and 48 months were correlated with Δ of the parameters analyzed. RESULTS We found a reduction of Δ height SDS from birth to 4 yr, with the main catch-up occurring during the first year, but only a slight, statistically insignificant reduction of Δ body mass index SDS during the observation period. Correlation coefficients were used to identify factors predicting postnatal catch-up growth. Both birth-weight difference (r=0.653; P=0.001) and Δ IGF-I in cord blood (r=0.613; P=0.007) were of similar predictive value. Variance analysis showed no significant difference in individual impact between these two parameters, although both correlated strongly with actual height. CONCLUSION We observed a gradual convergence in height but not body mass index up to the age of 4 yr between genetically identical twins with discordant birth weights. Both birth-weight and cord-blood IGF-I are predictive of subsequent catch-up growth.

TCF7L2 Polymorphism rs7903146 and Predisposition for Type 2 Diabetes Mellitus in Obese Children

Polymorphism RS7903146 in transcription factor 7-like2 gene ( TCF7L2) is associated with type 2-diabetes mellitus (T2DM) in adults. Concerned with predisposition for diabetes mellitus in obese children, we tested if risk genotypes TC and TT of rs7903146 are more common in obese children with increased homeostasis model assessment insulin resistance index (HOMA-IR) compared to obese controls with normal HOMA-IR. As exploratory analysis, we also calculated beta-cell function for these risk genotypes and measured glucagon-like peptide 1 (GLP-1) in a subgroup. The cohort was 401 obese children (BMI > 2SDS; 211 female; 59% presenting increased HOMA-IR) from two German outpatient obesity referral centers. Genotype distributions in patients presenting increased HOMA-IR (TT: 10.18%, CT: 35.65%, CC: 54.17%) and in patients with normal HOMA-IR (TT: 8.66%, CT: 42.67%, CC: 48.67%) provided no significant effect of these two risk genotypes (p > 0.2). Correction for possible confounders gender, age, pubertal stage, and BMI revealed no association with glucose metabolism parameters including GLP-1. However, exploratory HOMA-B% index was comparatively higher in TT-homozygotes (p=0.021) as compared to CC-homozygotes. We conclude that even though TT and CT genotypes were not higher in patients presenting elevated HOMA-IR, the higher HOMA-B% index in TT-homozygotes indicates TCF7L2 to be a susceptibility gene for the development of impaired glucose tolerance in obese children as demonstrated in several adult cohort studies.

Novel Acid-Labile Subunit (IGFALS) Mutation p.T145K (c.434C>A) in a Patient with ALS Deficiency, Normal Stature and Immunological Dysfunction

We report a novel missense mutation p.T145K in the insulin-like growth factor (IGF) acid-labile subunit (IGFALS) gene identified in a Turkish patient with normal growth, transient pancytopenic episodes and signs of immunological dysfunction. Because of recurrent cutaneous mycoses and absence of pubertal development until the age of 14.75 years we determined several endocrine parameters in order to rule out autoimmune-polyendocrine syndromes. Despite a normal height between the 25th and 50th percentile we found severely decreased IGF-1 and undetectably low IGFBP-3 levels. Laboratory signs of immunological dysfunction included reduced total lymphocyte count with diminished B and T helper cell fractions, decreased serum concentrations of IgM and IgG subclass 4, and elevated antinuclear antibody and anti-dsDNA titers as well as persistently high interleukin-2-receptor levels. Further endocrine work-up revealed elevated fasting insulin and undetectably low ALS serum levels, leading to the diagnosis of ALS deficiency. Sequencing of the coding region of the IGFALS gene showed a novel homozygous missense mutation (c.434C>A; p.T145K). Since immunological abnormalities have not been reported in more than 20 ALS-deficient patients so far and our patient was born to consanguineous parents, a second autosomal recessive defect is likely to underlie the immunological phenotype, although a causative role of IGFALS p.T145K cannot be entirely ruled out.

Association of COMT genotypes with S-COMT promoter methylation in growth-discordant monozygotic twins and healthy adults

BackgroundCatechol-O-Methyltransferase (COMT) plays a key role in dopamine and estrogen metabolism. Recently, COMT haplotypes rather than the single polymorphism Val158Met have been reported to underlie differences in protein expression by modulating mRNA secondary structure. So far, studies investigating the epigenetic variability of the S-COMT (soluble COMT) promoter region mainly focused on phenotypical aspects, and results have been controversial.MethodsWe assessed S-COMT promoter methylation in saliva and blood derived DNA with regard to early pre- and postnatal growth as well as to genotype for polymorphisms rs6269, rs4633, and rs4680 (Val158Met) in 20 monozygotic twin pairs (mean age 4 years), who were discordant for intrauterine development due to severe feto-fetal-transfusion syndrome. Methylation levels of two previously reported partially methylated cytosines were determined by the quantitative SIRPH (SNuPE- IP RP HPLC) assay.ResultsOverall, we observed a high variability of S-COMT promoter methylation, which did not correlate with individual differences in the pre- or postnatal growth pattern. Within the twin pairs however we noted a distinct similarity that could be linked to underlying COMT genotypes. This association was subsequently confirmed in a cohort of 93 unrelated adult controls. Interestingly, 158Val-alleles were found at both ends of the epigenotypical range, which is in accordance with a recently proposed model of COMT haplotypes corresponding to a continuum of phenotypical variability.ConclusionThe strong heritable component of S-COMT promoter methylation found in our study needs to be considered in future approaches that focus on interactions between COMT epigenotype and phenotype.

Growth hormone receptor d3-variant, insulin-like growth factor binding protein-1 –575G/A polymorphism and postnatal catch-up growth: Association with parameters of glucose homeostasis in former extremely low birth weight preterm infants

BACKGROUND Low birth weight predisposes to the development of insulin resistance. In addition to auxological parameters such as rapid catch-up growth, low IGFBP-1 serum levels in childhood have been linked to an increased risk of insulin resistance later in life. Concerning postnatal growth, we previously reported the GHRd3-variant to be associated with catch-up growth in preterm infants. In children born small for gestational age, a common IGFBP-1 promoter polymorphism -575G/A has been linked to IGFBP-1 serum levels and has been suggested to be an additional player in the interaction between the IGF-IGFBP-axis and metabolism. STUDY DESIGN We analyzed postnatal growth, metabolic parameters, and genotypes for the GHRd3-variant and IGFBP-1 -575G/A in 51 former extremely low birth weight preterm infants (mean age 5.9 years). RESULTS GHRd3 but not IGFBP-1 -575G/A was significantly associated with postnatal growth velocity. Catch-up growth, GHRd3, and IFGBP-1 -575G/A did not influence fasting insulin or HOMA-IR. However, we found significantly higher HbA1c and lower IGFBP-1 concentrations in GHRd3-carriers, a finding not seen with respect to IGFBP-1 -575G/A. Interestingly, HbA1c and IGFBP-1 levels also did not differ between children either with or without catch-up growth. CONCLUSIONS In addition to an association with catch-up growth, GHR exon 3 genotype significantly modulates HbA1c and IGFBP-1 concentrations in former ELBW infants. In order to confirm this observation and to clarify whether the GHRd3-variant might be considered as an independent modulator of the low birth weight infants risk to develop insulin resistance later in life, larger studies extending to later ages are required.