Cell Hypoplasia Research Articles

Pure Red Cell Aplasia and Myelofibrosis in B-cell Neoplasm

We describe an unusual case of B-cell neoplasm accompanied by pure red cell aplasia (PRCA) and myelofibrosis in a 67-year-old male presenting with severe anaemia. A few unclassified, myeloperoxidase-negative blastoid cells were seen on bone marrow aspiration, and erythroid cell hypoplasia and myelofibrosis on bone marrow biopsy. An autoimmune PRCA was suspected, as serum CH50, C3 and C4 levels were consistently low. Ciclosporin was effective in treating the anaemia, but anaemia returned when the drug was discontinued. Thirteen months later, the patient was admitted with pleural effusion and ascites that contained monoclonal CD19+ CD20+ immature blast cells with a complex karyotype, thought to be neoplastic B-cells. The unclassified blastoid cells seen earlier may therefore have been from the same origin. The patient deteriorated rapidly and died. Only one case of non-Hodgkin's lymphoma with PRCA and myelofibrosis has been reported previously. We discuss the possibility that dysregulated T-cells induced by neoplastic B-cells may have given rise to concomitant PRCA and myelofibrosis.

Reproductive Effects Of human Interferon-Alpha-2b Administration on Male Albino Mice Testes.An Experimental Study.

Background: Recombinant human interferon alpha (rh-IFN-α) is used therapeutically in malignant disorders and chronic hepatitis. The phenotypic effects of this drug at the structural levels on testicular tissue were hardly ever addressed. Hence, this work was designed in adult male albino mice to study the phenotypic effects of rh-INF-α-2b on testicular tissue as well as assessing its effects on serum testosterone and gonadotropins levels. Objective: This research was planned to through light on the effects of interferon-alpha- 2b (IFN-alpha-2b) on the hypothalamic-pituitary-testicular (HPT) axis of the adult male albino mice. Design: Experimental study. Setting: National Hepatology and Tropical Medicine Research Institute (NHTMRI). The study was conducted from November (2004) to February (2005). Materials and methods: Thirty sexually mature male mice were divided into three groups (10 mice in each group), namely: the control, the experimental and the recovery groups. Mice in the experimental and recovery groups were administered recombinant human interferon alpha intraperitoneally at a dose of 3000 U / mouse weekly for 12 weeks in a volume of 1.0-microliter isotonic normal saline, then animals in the recovery group were left to recover for a further period of two months. At the end of the experiment, serum concentrations of gonadotropins and testosterone were measured and then all animals were then sacrificed to study histopathologically the possible effects of interferon on the testicular tissue. Results: rh-IFN-α-2b induced remarkable decline in the serum levels of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in mice of the experimental group compared to the corresponding control and mice of recovery group. At the same time, testosterone was moderately increased in the experimental group, and then returned to its normal levels within 2 months after cessation of treatment. Histopathologically, in the experimental group, there were focal thickening of the basement membrane, degenerative changes and clumping of the germinal epithelial cells in the center of seminiferous tubules, partial desquamation of the germinal epithelium from basement membrane, reduction in the germ cell height, partial arrest of maturation and increased number of Sertoli cells. Increased number of Leydig’s cells and hypervascularity were detected in the interstitial spaces. In the recovery group, there was lessening of the germ cell hypoplasia manifested by restoration of spermatogenic cells and accidental disruption in the basement membrane. Most of the spermatogenic and Sertoli cells restored their polarity, height and maturation. Conclusion: our results suggest that rh-INF-α-2b temporally affects the hypothalamic- pituitary-testicular axis (HPT), both centrally and peripherally (at the testicular level), through the lessening of FSH, LH, raise of testosterone serum levels and direct phenotypic effect on the testicular tissue. 67

Can heterozygous intron mutations in the Luteinizing Hormone receptor gene cause delayed puberty?

Loss-of-function mutations of the LH receptor gene (Leydig cell hypoplasia) inactivate the receptor and give rise to primary amenorrhea and infertility in women and intersexual genitalia, hypospadias, micropenis and infertility in men. To evaluate if delayed puberty in boys and primary amenorrhoea in 46,XX girls can be due to partial Leydig cell hypoplasia we have studied the LH receptor gene in 35 patients. 8 girls were referred to pediatric endocrine outpatient clinics because of unexplained primary amenorrhoea and 27 boys because of extremely delayed puberty. The girls were 15.1–19.5 years old (mean 16.3 years) and showed normal breast development. The boys were 14,3–19,2 years old (mean 15.7 years) and presented with puberty Tanner stage 1 or 2 (testis volume below 5ml). In all male patients underlying diseases like chronic diseases or endocrine disorders especially hypogonadotropic hypogonadism by GnRH or Buserelin stimulation test were excluded. In all patients the protein-encoding part of all exons and the intron-exon boundaries were analyzed resulting in the identification of 7 new heterozygous LH receptor gene changes in 3 girls (+2 bp in Intron 5 (A to G); +18 bp in Intron 7 (C to T)) and in 5 boys (insertion of 18 AS, Exon 1; +3 bp in intron 1 (G to C); +5 bp Intron 5 (T to G); -8 bp Intron 8 (C to G); V553L in exon 11 (GTT to CTT). With exception of the alteration in intron 8 (-8 bp), all changes were not found in 300 control persons. On the other allele of the patients DNA no changes in the LH receptor gene were identified, indicating that the alterations identified in these patients cannot be linked free from doubt to LCH. Due to the fact that we have found in 2 not related girls presenting with primary amenorrhea the same heterozygous transition in intron 5 of the donor splicing site one may speculate that an aberrant splicing of one allele might affect the second allele. Further studies of more patients with delayed puberty and in vitro experiments with the mutant receptors will follow.

Novel insertion frameshift mutation of the LH receptor gene: problematic clinical distinction of Leydig cell hypoplasia from enzyme defects primarily affecting testosterone biosynthesis

Leydig cell hypoplasia (LCH) is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals and is caused by inactivating mutations of the LH receptor gene. The clinical and biochemical diagnostic parameters of LCH are not always specific and may therefore show significant overlap with other causes of insufficient testicular steroid biosynthesis. We have studied a 46,XY newborn with completely female external genitalia and palpable testes. Due to an increased basal serum ratio of androstenedione/testosterone, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD 3) deficiency was initially suspected. DNA analysis of the corresponding HSD17B3 gene, however, showed no abnormalities in the entire coding region. In contrast, direct sequencing of the LH receptor gene revealed a novel homozygous single nucleotide insertion in exon 11 (codon A589fs) producing a frame shift in the open reading frame predicting for premature termination of translation 17 amino acids downstream. From the genetic perspective, this mutation represents the first frame shift mutation in the LH receptor gene ever reported to date. From the clinical standpoint, LCH should always be considered in the differential diagnosis as steroid profiles may not be informative. Therefore, molecular genetic analysis should be warranted for androgen biosynthesis defects in all cases.

Hipoplasia das células de Leydig

The Leydig cell hypoplasia is a rare and well defined form of male pseudohermaphroditism with autosomal recessive inheritance pattern. An inadequate fetal testicular Leydig cell differentiation and, consequently a low androgenic production during intra uterine and post natal periods, result in absence or incomplete virilization in patients with 46,XY karyotype. These patients exhibit a wide clinical spectrum, ranging from complete female external genitalia to male external genital with micropenis, low serum testosterone levels associated with high LH levels. Inactivating mutations of the LH/hCG receptor gene have been identified in affected families in the last decade. However, the low frequency of inactivating mutations in this gene, and the lack of segregation of intragenic polymorphisms among affected members from families with typical phenotype of Leydig cell hypoplasia, suggest the genetic heterogeneity of this condition.

Murine Goblet Cell Hypoplasia during Eimeria pragensis Infection is Ameliorate by Clindamycin Treatment

The goblet cell (GC) and the intestinal mucus are important in preventing invasion of the mucosa by luminal microorgansisms. GC responses in the jejenum, cecum and colon of C57BL/6 mice during the course of infection with the large intestinal-tropic coccidian, Eimeria pragensis (E. pragensis), were investigated histologically. The numbers of large intestinal GCs (cecum and colon) gradually decreased (hypoplasia) in association with development of endogenous stages of parasite life cycle. The effect was transient and recovery of GC numbers was associated with resolution of coccidial infection. The jejunal GC numbers were not affected by E. pragensis infection. Clindamycin treatment in the infected mice reduced numbers of intracellular parasites and significantly increased the numbers of large intestinal GCs compared with untreated, infected mice.

Three New 46,XX Male Patients: A Clinical, Cytogenetic and Molecular Analysis

XX males range phenotypically from completely masculinised individuals to true hermaphrodites and include a subset of SRY negative patients. The correlation between genotype (SRY+/-) and phenotype is still unclear. To report three new patients with this rare condition, one of whom was diagnosed prenatally and another was SRY negative, and to verify in our patients whether the presence of SRY results in a more masculinised phenotype. We present two phenotypically normal XX male patients (10 and 13.5 years) and one 3.1 years old XX male with ambiguous external male genitalia Prader IV. The patients were diagnosed by clinical, hormonal, sonographic, genetic and histological criteria. Basal hormonal status was normal for phenotype but an excessive response to GnRH testing was noticed in the second patient together with insufficient hCG stimulation in all three patients. Pelvic ultrasound displayed male structures without Müllerian ducts; testicular biopsy, performed only in the intersex patient, showed Sertoli and Leydig cell hypoplasia. Chromosome analysis confirmed 46,XX karyotype. FISH analysis and molecular analysis by PCR were positive for Yp fragments/SRY gene on Xp in two patients and negative in the patient with ambiguous external genitalia. In our observation Y chromosome-specific material containing the SRY gene translocated to the X chromosome results in a completely masculinised phenotype. In the intersex patient, incomplete masculinisation without SRY suggests a mutation of one or more downstream non-Y testis-determining genes.

A Novel Double Mutation in the Luteinizing Hormone Receptor in a Kindred with Familial Leydig Cell Hypoplasia and Male Pseudohermaphroditism

We report a novel mutant of the luteinizing hormone receptor (LHR) in a case of familial Leydig cell hypoplasia and pseudohermaphrotidism. The proband was homozygous for two missense mutations, T1121C and C1175T, causing substitutions I374T and T392I. The molecular effects of the mutations were investigated by heterologous expression of the WT LHR, the double mutant LHR, or receptors with either the I374T or the T392I mutation, and measuring hormone binding and cAMP signaling. All mutant LHRs exhibited severe defects, including loss of ligand binding and cAMP production. Immunoblots showed little difference in protein levels between the WT and mutant receptors.

Targeted disruption of luteinizing hormone β-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) act on gonadal cells to promote steroidogenesis and gametogenesis. Clarifying the in vivo roles of LH and FSH permits a feasible approach to contraception involving selective blockade of gonadotropin action. One way to address these physiologically important problems is to generate mice with an isolated LH deficiency and compare them with existing FSH loss-of-function mice. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHbeta-subunit. LHbeta-null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, prominent Leydig cell hypoplasia, defects in expression of genes encoding steroid biosynthesis pathway enzymes, and reduced testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage, causing a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology demonstrates normal thecal layer, defects in folliculogenesis including many degenerating antral follicles, and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, because FSH levels were unaffected in null mice. Finally, both male and female null mice can be pharmacologically rescued by exogenous human chorionic gonadotropin, indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHbeta null mice represent a model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.

Leydig cell hypoplasia due to inactivation of luteinizing hormone receptor by a novel homozygous nonsense truncation mutation in the seventh transmembrane domain

Inactivating mutations in the LH receptor are the predominant cause for male pseudohermaphroditism in subjects with Leydig cell hypoplasia (LCH). The severity of the mutations, correlates with residual receptor activities. Here, we detail the clinical presentation of one subject with complete male pseudohermaphroditism and LCH. We identify within the proband and her similarly afflicted sibling a homozygous T to G transversion at nucleotide 1836 in exon 11 of the LH/ CGR gene. This causes conversion of a tyrosine codon into a stop codon at codon 612 in the seventh transmembrane domain, resulting in a truncated receptor that lacks a cytoplasmic tail. In vitro, in contrast to cells expressing a normal LHR, cells transfected with the mutant cDNA exhibit neither surface binding of radiolabeled hCG nor cAMP generation. In vitro expression under the control of the LHR signal peptide of either a wild type or mutant LHR-GFP fusion protein shows no differences in receptor cellular localization. In conclusion, the in vitro studies suggest that residues in the seventh transmembrane domain and cytoplasmic tail are important for receptor binding and activation without playing a major role in receptor cellular trafficking.

Value of In Vitro Models for the Assessment of Drug-Induced Haematotoxicity

BackgroundA new antipsychotic compound induced unexpected red cell hypoplasia (reticulocytopenia, red marrow hypoplasia) in rats dosed orally for 7days. Materials and methodsSince an erythropoietin-mediated pathogenesis was excluded, in vitro tests on rat and human bone marrow cells were performed with measurement of formation of late erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units after incubation with the drug. CFU-E together with growth factors were cultured for 2days (rat) or 7days (human) and CFU-GM was cultured for 7days (rat) or 10days (human). ResultsThe drug induced inhibition of erythroid progenitors and myeloid progenitors for both species from 3×10−5 mol/L, with the concentration inhibiting the growth of 50% (IC50) consistent with drug plasma levels measured in rats. ConclusionThese cloning assays on rat bone-marrow cells were shown to be adequate models for determining the haematotoxicity of the agent and to be predictive of human toxicity. With only a small amount of compound required, they can be used as screening tools to detect haematotoxic potential of candidate drugs.

Detection of rare Leydig cell hypoplasia in somatic cell cloned male piglets

In this investigation, 22 cloned male piglets were obtained by male fetal fibroblast-cell-derived nuclear transfer. Eighteen of the cloned animals died. The two cell lines did not differ significantly with regard to efficiency of live piglet production. The gross anatomy of the testes of male piglets that died was normal. However, one piglet displayed Leydig cell hypoplasia (LCH). No anatomical defects were detected in the testes of other cloned male piglets. TUNEL analysis of the testis with LCH revealed significant apoptosis in the Leydig cells, while apoptosis was rarely detected in Sertoli cells and spermatogonia. In contrast, testes from the remaining 17 piglets that died appeared normal in size, and their Sertoli and Leydig cell numbers were comparable to those in control piglet testes. Although cloned piglets were derived from fibroblasts obtained from the same fetus, phenotypic instability between cells used for the production of somatic cell cloned piglets suggests that abnormalities in male cloned piglets are caused not by technical problems and/or reprogramming effects, but rather by epigenetically and/or genetically damaged cell-specific effects.

Congenital Pulmonary Airway Malformation (Congenital Cystic Adenomatoid Malformation) with Multiple Extrapulmonary Anomalies: Autopsy Report of a Fetus at 19 Weeks of Gestation

Congenital pulmonary airway malformation, or congenital cystic adenomatoid malformation, is postulated to be a disorder of pulmonary airway morphogenesis and encompasses 5 different types with distinct levels or stages of tracheobronchial development. We present a unique case of type 2 congenital pulmonary airway malformation with a previously undocumented combination of multiple extrapulmonary anomalies, featuring ipsilateral multicystic renal dysgenesis, contralateral renal agenesis, and ovarian germ cell hypoplasia, diagnosed in a 19-week gestational age fetus by autopsy. Epithelial cells comprising the pulmonary lesions were positive for thyroid transcription factor-1, surfactant protein-B, and cytokeratin-7 but negative for cytokeratin-20 immunostainings, with the pattern seen in normal terminal bronchioles. Chromosomal analysis showed a normal female karyotype, despite a high estimated risk for Down syndrome suggested by the low maternal serum alpha-fetoprotein level.

65RARE AND OFTEN UNRECOGNIZED CEREBROMENINGITIS AND HEPATOPNEUMONIC CONGESTION ARE MAJOR CAUSES OF SUDDEN DEATH IN CLONED MALE PIGLETS

In this study, we investigated the relationship between the sex of cloned pigs and sudden infant death syndrome (SIDS). Three cell lines (2 male and 1 female) were obtained from F1 fetuses derived from 3 different dams (Yorkshire) inseminated by the same sire (Landrace); one female fibroblast cell line was obtained from a Duroc-strain fetus acquired from a slaughterhouse, the age of the fetus unknown. The fetal fibroblast cells were cultured in DMEM supplemented with 10% fetal bovine serum under 5% CO2 in air at 37°C. For NT, we used the 4 cell lines described above. All 37 cloned piglets derived from 10 pregnant recipients were alive at term and began breathing readily. Of those piglets born, 18/22 males and 5/15 females died within 2 months of age. A total of 350 paraffin blocks from 18 deceased cloned male piglets and 90 paraffin blocks from six age-matched normal control piglets were prepared from the midbrain, medulla oblongata, liver, lung, kidney, spleen, small and large intestine, thymus, uterus, placenta, ovary, testis, skin, and skeletal muscle. We found that the birth weights of male clones were 57% lower than those of control age- and sex-matched piglets. Piglets with low birth weight (<900g) had more than twice the risk of SIDS relative to those piglets weighing more than 1000g. The low birth weights of the cloned male piglets (0.84±0.05kg) were not merely an artifact, as the average birth weights of cloned female piglets (1.47±0.09kg) were not lower than weights of piglets produced by AI-derived control female piglets (1.36±0.12kg). An initial examination of brain samples from 18 cloned male piglets that died soon after birth identified seven piglets with meningitis characterized by severe neutrophilic inflammation in the temporal brain lobes (38.8%, 7/18). We verified meningitis when more than 1000 neutrophils were counted per cubic millimeter of tissue. Next, we found hepatopneumonic congestion (16.6%, 3/18). The deceased male clones with meningitis showed extensive neuronal cell death and blood-brain barrier damage, whereas cloned piglets with congestion had fewer and larger alveolar air sacs. Extensive alveolar cell death, especially of pneumocytes and the bronchial epithelium, was confirmed by TUNEL assay. Lung and liver congestion may be caused by a slowly flowing blood stream from heart, resulting in CO2 and O2 exchange problems. Although the gross anatomy of the cloned male piglets was normal, they were associated with other severe handicaps, the commonest being leg abnormality which occurred in 33% (6/18) of dead male cloned piglets followed by Leydig cell hypoplasia and short face. Even though 4 of 41 AI-derived control piglets died within 1 week after birth, we could not find any anomalies in them. Thus, the present study suggests that our data might reflect sex difference but not cell type difference, and that death of the cloned male piglets might be caused by risk factors of sudden infant death syndrome such as low birth weight and multiple organ failure in conjunction with hepatopneumonic congestion and cerebromeningitis.

Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia.

It is unclear whether or not testicular sperm extraction (TESE) should be repeated for patients in whom no sperm were found during their first TESE attempt. The outcome of repeated TESE was evaluated in patients with non-obstructive azoospermia (NOA) after failing to obtain sperm in their first extraction attempt, or having used all available cryopreserved testicular tissue. Out of 83 patients with NOA, patients repeated TESE two (n = 22), three (n = 8), four (n = 6) and five (n = 3) times. Distribution of main testicular histology included germ cell aplasia (55%), maturation arrest (29%) and germ cell hypoplasia (16%). The first TESE yielded mature sperm for ICSI in 39% of patients (sp+), and failed in the remaining 61% (sp-). A second TESE yielded mature sperm in 1/4 from the sp- group and in 16/18 from the sp+ group. At the third, fourth and fifth trials, 8/8, 5/6 and 3/3 of the original sp+ patients were sp+ again respectively. Compared with the outcome of the first trial, all further trials did not differ statistically in the rate of fertilization (54 versus 49%), implantation (9.5 versus 5.4%), or clinical pregnancy/cycle (19 versus 15%). No pregnancies were achieved among the three patients after their fifth TESE. Pregnancies occurred in all histological groups, except maturation arrest. The outcome of repeated TESE cycles, up to the fourth trial, justifies the procedure.

Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency

Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency

Leydig cell hypoplasia: cases with new mutations, new polymorphisms and cases without mutations in the luteinizing hormone receptor gene.

Defective male sex differentiation in patients with hypoplasia of Leydig cells (LCH) is caused by deficient LH receptor signal transduction. To further investigate the variety of LH receptor gene mutations present in LCH patients and their influence on the phenotype, we examined 10 nonrelated patients with the clinical presentation of LCH. Ten patients with a clinical phenotype of LCH were analysed for mutations in the complete coding region of the LH receptor gene. Exons 1-10 and two overlapping fragments of exon 11 of the LH receptor gene including all intron-exon boundaries were amplified by polymerase chain reaction and sequenced. To screen for frequencies of DNA changes, mutation analysis was performed on 45-59 healthy persons using denaturation high-performance liquid chromatography. Six new DNA alterations were identified. Three of them appear to be new polymorphisms. A G to C change at the 28th nucleotide of intron 1 on one allele and a heterozygous CGA to CAA transition at codon 124 (R124Q) were found. Both findings in these two patients are polymorphisms that occur with a frequency of 17% and 1.7%, respectively. A silent heterozygous CTA to TTA change at codon 204 was identified. In a patient with micropenis, the analysis revealed a homozygous missense mutation at codon 625 (I625K). As reported previously, this alteration significantly impaired signal transduction and explains the partial phenotype. Finally, in one compound heterozygous patient, two different mutations were discovered. At the polymorphic site in exon 1, a 27-bp insertion (CTG)2 AAG (CTG)5 CAG and a premature stop codon in the transmembrane segment 4 (W491*) were found. Both mutations disrupt signal transduction and explain the complete phenotype of this patient. In five patients, no DNA alterations could be identified. Three mutations (33 bp insertion in exon 1; W491* and I625K) were identified that explain the phenotype in two patients. In addition, most of the patients with the clinical phenotype of LCH did not have causative mutations, suggesting that changes in other regions of the LH receptor gene, such as the large introns or the promoter region, may be responsible for the majority of cases. Alternatively, the displayed phenotype may be the result of other genetic defects. Our work further underscores the importance of thorough clinical analysis of patients before molecular analysis of a particular gene is performed.

LH receptor defects.

In this article the role of LH receptor gene mutations in patients with aberrant sex differentiation is discussed. In a dominant autosomal familial form of precocious puberty in boys (familial male-limited precocious puberty) LH receptor gene mutations have been identified. These single amino acid changes, mostly found in the sixth transmembrane helix and the third intracellular loop of the transmembrane domain of the LH receptor, cause constitutive activation of LH receptor protein without the hormone present, resulting in precocious production of testosterone by the testicular Leydig cells. The large number of activating LH receptor mutations have allowed more precise molecular modeling of the LH receptor protein. In a rare hereditary form of 46,XY male pseudohermaphroditism known as Leydig cell hypoplasia, LH receptor gene mutations have been identified that completely or partially inactivate the LH receptor protein. Large gene deletions cause complete absence of the LH receptor protein, whereas other, more subtle missense mutations prevent the receptor from assuming an active conformation.

Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency

Over the past 5 yr several inactivating mutations in the LH receptor gene have been demonstrated to cause Leydig cell hypoplasia, a rare autosomal recessive form of male pseudohermaphroditism. Here, we report the identification of two new LH receptor mutations in a compound heterozygous case of complete Leydig hypoplasia and determine the cause of the signaling deficiency at a molecular level. On the paternal allele of the patient we identified in codon 343 a T to A transversion that changes a conserved cysteine in the hinge region of the receptor to serine (C343S); on the maternal allele a T to C transition causes another conserved cysteine at codon 543 in trans-membrane segment 5 to be altered to arginine (C543R). Both of these mutant receptors are completely devoid of hormone-induced cAMP reporter gene activation. Using Western blotting of expressed LH receptor protein with a hemagglutinin tag, we further show that despite complete absence of total and cell surface hormone binding, protein levels of both mutant LH receptors are only moderately affected. The expression and study of enhanced green fluorescent protein-tagged receptors confirmed this view and further indicated that initial translocation to the endoplasmic reticulum of these mutant receptors is normal. After that, however, translocation is halted or misrouted, and as a result, neither mutant ever reaches the cell surface, and they cannot bind hormone. This lack of processing is also indicated by reduced presence of an 80-kDa protein, the only N-linked glycosylated protein in the LH receptor protein profile. Thus, complete lack of signaling by the identified mutant LH receptors is caused by insufficient processing from the endoplasmic reticulum to the cell surface and results in complete Leydig cell hypoplasia in this patient.