Aleksandra Rojek

NR0B1 ( DAX1 ) mutations in patients affected by congenital adrenal hypoplasia with growth hormone deficiency as a new finding

X-linked adrenal hypoplasia congenita (AHC,OMIM#300200) is a rare disorder of the adrenal cortex caused bymutations in the NR0B1 (DAX1) (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene (OMIM*300473). NR0B1 (DAX1) is located on the short arm of the X chromosome (Xp21.3-p21.2) and encodes for an orphan nuclear hormone receptor (transcription factor) (Zanaria et al. 1994). NR0B1 (DAX1) is expressed in the adrenals, gonads, hypothalamus, and pituitary gland, and it is responsible for controlling the development and function of these tissues. NR0B1 (DAX1) protein functions as a dominant negative regulator (transcriptional repressor) in the hypothalamic– pituitary–adrenal–gonadal axis. It regulates the expression of other proteins involved in the adrenal steroidogenesis pathway, such as steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), P450scc and 3β-hydroxysteroid dehydrogenase (Lalli et al. 2000; Iyer and McCabe 2004). Almost 200 mutations in theNR0B1 (DAX1) gene have been identified to date (The Human Gene Mutation Database—data for 10.08.11) (http://www.hgmd.org). The most commonly found mutations in AHC patients are frameshift and nonsense mutations, which are located throughout the entire length of the NR0B1 (DAX1) gene. Missense mutations are not so common and are located mainly in the region that encodes for a ligandbinding domain at the C-terminus of the NR0B1 (DAX1) protein (Achermann et al. 2001a). Some patients have more complex X-chromosome rearrangements that include the deletion of larger parts of the X chromosome, including NR0B1 (DAX1), DMD (dystrophin), GK (glycerol kinase) and IL1RAPL (interleukin 1 receptor accessory protein-like) genes (Choi et al. 2005). AHC symptoms are noticed very early in postnatal life. The disorder is characterized by salt-wasting syndrome, hypoglycemia in early infancy or childhood, poor weight gain, vomiting, prolonged neonatal jaundice, and skin hyperpigmentation. In laboratory tests, hyponatremia, hyperkalemia, low levels of cortisol, aldosterone, and adrenal androgens are found, in contrast to high levels of adrenocorticotropic hormone (ACTH) and plasma renin activity (Achermann et al. 2000; Nakae et al. 1997; Wiltshire et al. 2001; Verrijn Stuart et al. 2007). Very frequently, patients are admitted to hospital in a life-threatening condition. AHC is lethal unless appropriate steroid replacement therapy is provided to these patients. Most patients with AHC also suffer from hypogonadotropic hypogonadism, since mutations in the NR0B1 (DAX1) gene A. Rojek (*) :M. Niedziela 2nd Chair of Pediatrics, Department of Pediatric Endocrinology and Rheumatology, Molecular Endocrinology Laboratory, Poznan University of Medical Sciences, 27/33 Szpitalna Street, 60-572 Poznan, Poland e-mail: aleksandra.rojek@gmail.com

A novel mutation in the NR0B1 (DAX1) gene in a large family with two boys affected by congenital adrenal hypoplasia

OBJECTIVE: X-linked Adrenal Hypoplasia Congenita (AHC) is a rare disorder caused by mutations in NR0B1 (DAX1) gene. DESIGN: We present two boys (cousins) with AHC who came to our attention at the age of 10 days and 15 days, respectively, in a life-threatening state. Laboratory studies in their neonatal periods showed hyponatremia and hyperkalemia. Primary adrenal insufficiency was confirmed, with severely low serum cortisol levels and high plasma ACTH levels. Hydrocortisone therapy with additional saline and glucose infusions were started immediately. Two exons of the NR0B1 (DAX1) gene were amplified using PCR and directly sequenced. RESULTS: Molecular analysis of the NR0B1 (DAX1) gene revealed a novel mutation, c.315G > A (p.W105X) in exon 1, resulting in the formation of a premature stop codon. Further studies showed that mothers, the maternal grandmother and two of six maternal great aunts were heterozygotes for the mutation. However, the mutation was absent in the maternal great-grandmother. CONCLUSIONS: We show that NR0B1 (DAX1) gene analysis is of great importance for the confirmation of the clinical diagnosis of AHC and highlights the role of genetic counseling for families of AHC patients. The absence of a somatic mutation in the great-grandmother suggests gonadal mosaicism as the mechanism for transmission of the NR0B1 (DAX1) mutation in this family.

Treatment of cartilage-hair hypoplasia with recombinant human growth hormone.

Cartilage–hair hypoplasia (CHH) is an autosomal recessive disorder characterized by short stature, hypoplastic hair and humoral immunity disorders. It is a mutation in the RMRP gene, located on chromosome 9p13.3, that leads to CHH. There is no special treatment for short stature in CHH. The efficacy and safety of recombinant human growth hormone (rhGH) therapy in CHH is still under discussion. The present study describes the case of a girl with CHH who was treated with rhGH. The rhGH treatment had a significant effect on the height gain: the height SD score was changed from −4. to −2.98 after 4 years 7 months of treatment. rhGH therapy should be considered as a treatment modality for CHH, and insulin‐like growth factor (IGF)‐1 and IGF‐binding protein 3 concentrations should be closely monitored, particularly because of the increased cancer risk that is a characteristic feature of CHH.

Adrenal function and MC1R gene analysis in a prepubertal girl with generalized hyperpigmentation: case report

Generalized hyperpigmentation (GHPT) of the skin may occur as a primary defect of pigmentation or in combination with other variable manifestations. It is visible in a number of diseases such as Addisons disease (AD), haemochromatosis, porphyria cutanea tarda, scleroderma and neurofibromatosis, but it can also be associated with malignancy and the use of chemotherapeutics or it can be related to acanthosis nigricans in insulin resistance. Skin pigmentation depends on the differences in the amount, type and distribution of melanin produced during melanogenesis in skin melanocytes [1] and remains under the genetic control of more than 120 genes [2]. The most important one is the melanocortin 1 receptor (MC1R) gene [3] (OMIM ID: 155555) located on chromosome 16q24.3 and encoding for a 317-amino-acid G-protein coupled receptor. The MC1R receptor binds α-melanocyte-stimulating hormone (α-MSH) resulting in the activation of adenylyl cyclase, which produces cyclic adenosine monophosphate (cAMP). The increased cAMP concentration activates various intracellular molecular pathways, promotes melanin synthesis and increases the eumelanin to pheomelanin ratio [4]. MC1R receptor also binds ACTH, in this way contributing to the GHPT in AD. Upregulation of MC1R gene expression by UV radiation and α-MSH leads to enhancement of melanogenesis and melanin synthesis induction. Loss-of-function mutations in the MC1R gene are associated with fair skin, poor tanning, propensity to freckles and increased skin cancer risk due to a decrease in eumelanin synthesis and subsequently impaired protection against UV radiation [5–7]. To our knowledge, to date, no data are available considering gain-of-function mutations in the human MC1R gene which could lead to a constant activation of the MC1R receptor and subsequently cause GHPT. We present the case of a patient with a primary type of progressive GHPT in whom AD was suspected. An 11-year-old prepubertal girl with GHPT (Figures 1A-​-C)C) was born at term with normal birth weight and height and was first brought to our hospital at the age of 3 years with a suspicion of AD. She had a diffuse grey-brownish discoloration of the skin present since birth. Over the first few years of life she developed symmetrical hyperpigmentation most pronounced on her trunk and neck. Later, hyperpigmentation began to affect her hands and feet, and finally the whole body – sparing only the cheeks and finger tips. Her skin was very dry and atopic, and scars were not hyperpigmented. Her toenails as well as fingernails had no major alterations. The girl also experienced chronic diarrhoea, once a month on average, that started at the age of 10-12 months, always considered as allergic diarrhoea. She did not have any history of repeating infections. Her mother also reported a strong ammonia odour of the girls urine. The remainder of the physical examination, mental development, abdominal ultrasound and magnetic resonance imaging (MRI) of the head were unremarkable. The girls height and weight both are on the 25th percentile and the onset of puberty was noticed (thelarche Tanner 2). Metabolic diseases were ruled out by serum and urine analysis. Ophthalmologic and neurologic examinations showed no abnormalities. Familial pedigree analysis did not show any similar cases in the patients family. The adrenocorticotropin (ACTH) stimulation test was performed several times by intravenous injection of synthetic ACTH (Table I). Cortisol levels were normal after ACTH stimulation, thus excluding AD. The patient acquired a chickenpox infection which presented as small white, unpigmented spots on the whole body, but hyperpigmentation was still present, especially on the girls neck and trunk. A histopathological examination of a skin biopsy from the buttock revealed a strong pigmentation of the epidermal basal layer and melanin aggregates within melanophages of the stratum papillare. The number of melanocytes within the epidermis was unchanged and melanosis diffusa congenita was diagnosed (OMIM ID: 145250). Figure 1 The proband at the age of 11 manifesting a clinical feature of generalized hyperpigmentation (A) with the most intensive pigmentation on her trunk (A, B), neck (B), extremities (A, C) and hands (C) Table I Laboratory tests in blood samples (abnormal values are given in bold). A single measurement of oestradiol was elevated at the age of 3 years but with no breast development. Slightly elevated ACTH level at the age of 8 years was probably a result of the ... Written informed consent was obtained from parents and the study was approved by the local Ethics Committee of Poznan University of Medical Sciences. Blood samples were collected and frozen at –20°C until analysis. All laboratory tests were measured in the Central Laboratory of K. Jonschers Clinical Hospital of the University using commercial kits. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), parathyroid hormone (PTH), prolactin, estradiol, free thyroxine (fT4) and insulin were assayed by MEIA, thyroid peroxidase antibody (TPO-Ab), 21-hydroxylase-antibody (21-hydroxylase-Ab), cortisol, aldosterone, dehydroepiandrosterone sulphate (DHEA-S) and C-peptide by RIA, and adrenocorticotropic hormone (ACTH) IRMA methods. Genomic DNA was isolated from peripheral blood leukocytes using QIAamp® DNA Blood Mini Kit (QIAGEN). Primer sequences used to amplify the MC1R gene as well as polymerase chain reaction (PCR) conditions are available on request. The samples were subjected to direct sequencing using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and an ABI Prism 3130XL Genetic Analyzer (Applied Biosystems). We did not find any mutations within the whole MC1R gene. The most likely diagnosis of the patient with GHPT is primary adrenal insufficiency, subsequent ACTH excess and overstimulation of MC1R in the skin. Recurrent episodes of diarrhoea in the patient could suggest an adrenal crisis but several adrenal function tests, also performed during such episodes, excluded adrenal failure. Although we did not find any mutations in the coding region of the MC1R gene, or in the untranslated regions or the promoter region, it is possible that disorders in other genes are responsible for the skin pigmentation phenotype in our patient. Branicki et al. showed that HERC2 rs12913832 may be one of the genes responsible for masking the effect of MC1R polymorphisms and significantly affect the function of the MC1R receptor [8]. The phenotype of our patient was characterized by diffuse GHPT, which varied slightly in intensity in different parts of her body. Cafe-au-lait macules or larger hypopigmented macules were not present. This is contrary to patients previously described in the literature [9]. Therefore, we concluded that the pathogenesis is restricted only to melanogenesis-related functions in the skin. Familial progressive hyperpigmentation (FPH, OMIM ID: 145250) is a rare congenital diffuse hyperpigmentation disorder affecting the human skin. Familial progressive hyperpigmentation is inherited in an autosomally dominant [9] or recessive [10] manner and is characterized by patches of hyperpigmentation present at birth or in early infancy that increase in size and also in number with age. Ultimately, a large percentage of the skin becomes hyperpigmented. These skin changes always occur on the face, neck, trunk, limbs, lips, oral mucosa and on the palms and soles, but this is not the case for our patient, whose lips, oral mucosa and conjunctiva were not hyperpigmented. Histological examination reveals an increase in the amount of melanin pigment found throughout the epidermis, specifically within the stratum corneum. The skin is the only affected organ and patients do not normally suffer from any other systemic disease [11, 12]. The molecular background of the disease still remains unknown. Zhang et al. examined a three-generation Chinese family consisting of 17 individuals, 6 of whom were affected, including 3 males and 3 females. Contrary to the previous study and our patient, the first onset of the disease in this family was at the age of 5 years, not at birth. All 6 affected members had typical clinical manifestations of FPH. They had normal mental abilities and no systemic disease such as gastrointestinal, liver or kidney ailments. The pedigree of the family showed an autosomal dominant pattern of FPH inheritance [13]. This study gives new potential insight into the plausible bipotential role of ACTH in both adrenal stimulation via MC2R and in skin pigmentation via MC1R, both transduction processes effectively activated at different plasma concentrations of ACTH. Because there were no disease-causing gain-of-function mutations in the MC1R gene, we concluded that this gene is not responsible for the hyperpigmented phenotype in our patient. Further studies of other candidate genes crucial for human skin pigmentation as well as genetic analysis of the selected SNP polymorphisms will help us to understand the molecular background of the GHPT disorder present in our case.

Molecular Detection and Incidence of Y Chromosomal Material in Patients with Turner Syndrome

The presence of a Y chromosome in patients with Turner syndrome (TS) is a risk factor for the development of gonadal tumor and/or virilization. With conventional cytogenetic analysis, some cells containing a Y chromosome can be missed. The aim of this study was to determine the presence and incidence of Y chromosome-derived material in TS patients using PCR and the markers SRY, DYZ1, DYZ3, DYS132, ZFY, and TSPY. Fifty-five TS patients (aged 5.5-26.75 years) were analyzed. A total of 17/55 (30.9%) were Y-positive, but only 7/17 had a Y chromosome in their karyotype and underwent gonadectomy. In 2 of these patients (28.6%), histopathologic examination revealed gonadoblastoma and dysgerminoma, respectively. In 8 patients in the studied group (8/55; 14.5%), the TSPY gene was detected, and the SRY gene (or a fragment) was identified in 9(3)/55 patients. No coding region mutations were observed in these SRY-positive patients. In conclusion, we have shown a high prevalence of Y chromosomal material in TS. Y markers were also observed in patients who had no Y chromosome in their karyotype, and PCR is very precise in detecting the presence of genetic material from the Y chromosome. Further follow-up of these Y-positive TS patients is mandatory.

X-Linked Adrenal Hypoplasia Congenita in a Boy due to a Novel Deletion of the Entire NR0B1 (DAX1) and MAGEB1–4 Genes

X-linked Adrenal Hypoplasia Congenita (AHC) is caused by deletions or point mutations in the NR0B1 (DAX1) gene. We present a boy with AHC who came at the age of 25 days in a severe state due to prolonged vomiting and progressive dehydration. Laboratory studies showed prominent hyponatremia and hyperkaliemia but not hypoglycemia. Primary adrenal insufficiency was confirmed with low serum cortisol levels and high plasma ACTH levels. Hydrocortisone therapy combined with saline and glucose infusions was started immediately after blood collection. Two exons of the NR0B1 (DAX1) gene were impossible to amplify using the standard PCR method. Array CGH was used to confirm the putative copy-number variation of NR0B1 (DAX1) revealing a novel hemizygous deletion encompassing the entire NR0B1 (DAX1) gene together with the MAGEB genes. This genetic defect was also present in heterozygosity in the patients mother. We show that NR0B1 (DAX1) gene analysis is important for confirmation of AHC diagnosis and highlights the role of genetic counseling in families with AHC patients, particularly those with X chromosome microdeletions, covering more than NR0B1 (DAX1) alone. We hope that further clinical follow-up of this patient and his family will shed a new light on the role of MAGEB genes.

Insulin Receptor and its Relationship with Different Forms of Insulin Resistance

Summary Insulin plays an important role in maintaining the whole organism’s homeostasis. The presence of insulin receptors in all vertebrates and invertebrates cells reflects the diversity of regulatory processes in which this hormone is involved. Furthermore, many different factors may influence the level of insulin receptor expression. These factors include e.g. the sole insulin or stage of development. Mutations in the receptor may lead to the development of insulin resistance. These mutations differ in the level of severity and are frequently associated with diabetes mellitus, hypertension, cardiovascular disorders, heart failure, metabolic syndrome and infertility in women. More than 50 mutations in insulin receptor gene have already been characterized. These mutations are associated with rare forms of insulin resistance like leprechaunism, insulin resistance type A or Rabson-Mendenhall syndrome. Molecular analysis of insulin receptor gene may lead to a better understanding of molecular mechanisms underlying various types of insulin resistance and help to develop more efficient treatment.