Evaluation of a Novel Immunochromatographic Assay Using Monoclonal Antibodies against the Matrix Protein of Human Metapneumovirus.

BACKGROUND: The aim of this study was to determine the sensitivity and specificity of a novel immunochromatographic (IC) assay (APD1806) using monoclonal antibodies against the matrix (M) protein of human metapneumovirus (hMPV) for detection of hMPV from nasopharyngeal swab samples based on the results of real-time RT-PCR. METHODS: Nasopharyngeal swab samples taken from 189 patients aged 0 - 5 years who were suspected of having respiratory tract infections associated with hMPV were used in this study. The samples were tested both by the IC assay and by real-time RT-PCR for detection of hMPV. RESULTS: The sensitivity and specificity of the IC assay for detection of hMPV were 88.8% (95/107) and 92.7% (76/82), respectively. CONCLUSIONS: The IC assay using monoclonal antibodies against the M protein of hMPV is an accurate and fast assay that is suitable as a diagnostic tool for hMPV infection. The optimal timing of the IC assay is 12 hours or more after the onset of fever due to hMPV infection.

A novel heterozygous mutation of the WFS1 gene leading to constitutive endoplasmic reticulum stress is the cause of Wolfram syndrome.

BACKGROUND: Wolfram syndrome (WS) is a disorder characterized by the association of insulin-dependent diabetes mellitus (DM), diabetes insipidus, deafness, and optic nerve atrophy. WS is caused by WFS1 mutations encoding WFS1 protein expressed in endoplasmic reticulum (ER). During ER protein synthesis, misfolded and unfolded proteins accumulate, known as "ER stress". This is attenuated by the unfolded protein response (UPR), which recovers and maintains ER functions. Because WFS1 is a UPR component, mutant WFS1 might cause unresolvable ER stress conditions and cell apoptosis, the major causes underlying WS symptoms. We encountered an 11-month-old Japanese female WS patient with insulin-dependent DM, congenital cataract and severe bilateral hearing loss. OBJECTIVE: Analyze the WFS1 and functional consequence of the patient WFS1 in vitro. RESULTS: The patient WFS1 contained a heterozygous 4 amino acid in-frame deletion (p.N325_I328del). Her mutant WFS1 increased GRP78 and ATF6α promoter activities in the absence of thapsigargin, indicating constitutive ER stress and nuclear factor of activated T-cell reporter activity, reflecting elevated cytosolic Ca2+ signals. Mutant transfection into cells reduced mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase 2b (SERCA2b) compared with wild type. Because SERCA2b is required for ER and cytoplasmic Ca2+ homeostasis, decreased SERCA2b expression might affect ER Ca2+ efflux, causing cell apoptosis. CONCLUSION: A novel heterozygous mutation of WFS1 induced constitutive ER stress through ATF6α activation and ER Ca2+ efflux, resulting in cell apoptosis. These results provide new insights into the roles of WFS1 in UPR and mechanism of monogenic DM.

Spondyloepiphyseal dysplasia congenita caused by double heterozygous mutations in COL2A1.

Spondyloepiphyseal dysplasia congenita (SEDC) is a group of rare inherited chondrodysplasias characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. SEDC is usually caused by substitution of glycine residue with another amino acid in the triple helical domains of alpha 1 chains, which consist of type II collagen (COL2A1). Herein, we describe a unique case of SEDC with mild coxa vara (SEDC-M) caused by double de novo COL2A1 mutations located on the same allele. One mutation, p.G504S, was previously described in patients with SEDC, whereas the other, p.G612A, was a novel mutation; both were located in the triple helical domain. Neither mutation was identified in the parents and appeared to be de novo. To the best of our knowledge, this is the first study involving a patient with a type II collagenopathy with two COL2A1 mutations on the same allele. The case was characterized by a more severe phenotype compared with previously reported cases involving a single p.G504S mutation, which may have been the result of the double mutation.

A Japanese family with nonautoimmune hyperthyroidism caused by a novel heterozygous thyrotropin receptor gene mutation.

BACKGROUND: Hyperthyroidism caused by activating mutations of the thyrotropin receptor gene (TSHR) is rare in the pediatric population. METHODS: We found a Japanese family with hyperthyroidism without autoantibody. DNA sequence analysis of TSHR was undertaken in this family. The functional consequences for the Gs-adenylyl cyclase and Gq/11-phospholipase C signaling pathways and cell surface expression of receptors were determined in vitro using transiently transfected human embryonic kidney 293 cells. RESULTS: We identified a heterozygous mutation (M453R) in exon 10 of TSHR. In this family, this mutation was found in all individuals who exhibited hyperthyroidism. The results showed that this mutation resulted in constitutive activation of the Gs-adenylyl cyclase system. However, this mutation also caused a reduction in the activation capacity of the Gq/11-phospholipase C pathway, compared with the wild type. CONCLUSION: We demonstrate that the M453R mutation is the cause of nonautoimmune hyperthyroidism.

A novel mutation of IGSF1 in a Japanese patient of congenital central hypothyroidism without macroorchidism.

Congenital central hypothyroidism (C-CH) is a rare disease known to be caused by mutations of the genes encoding TSH ß or the TRH receptor gene, although the cause of the disease in a number of patients has not yet been clarified. Recently, mutations and deletions of the immunoglobulin superfamily member 1 (IGSF1) gene have been reported to be the cause of C-CH. Here we report a Japanese male patient with C-CH due to a novel IGSF1 mutation. He was detected by neonatal mass screening of simultaneous TSH and free T4 measurements and levothyroxine was initiated. At 6 years of age he underwent ¹²³I scintigraphy after levothyroxine treatment had been discontinued for one month and his thyroid and pituitary function were evaluated. Since TSH and PRL responses after TRH stimulation were low, his diagnosis of C-CH was confirmed. During follow up, whereas onset of his puberty was delayed, his secondary sex characterization completed at 17 years old. In this patient we analyzed IGSF1 and TRHR. As results, we identified a novel insertion mutation in IGSF1 (c.3528-3529insC), resulting in a premature stop codon (p.Pro1082Trpfs39X). In conclusion, we identified a novel mutation of IGSF1 in a Japanese male patient with C-CH.

Two novel mutations of the CYP11B2 gene in a Japanese patient with aldosterone deficiency type 1.

Isolated hypoaldosteronism is a rare and occasionally life-threatening cause of salt wasting in infancy. A 2-month-old Japanese boy of unrelated parents was examined for failure to thrive and poor weight gain. Laboratory findings were hyponatremia, hyperkalemia, high plasma renin and low aldosterone levels. Spot urine analysis by gas chromatography-mass spectrometry (GC-MS) showed that urinary excretion of corticosterone metabolites was elevated. Whereas excretion of 18-hydroxycortricosterone metabolites was within the normal range, excretion of aldosterone metabolites was undetectable. The patient was therefore suspected to have aldosterone synthase deficiency type 1. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (CYP11B2), showed that the patient was a compound heterozygote for c.168G>A, p.W56X in exon 1 and c.1149C>T, p.R384X in exon 7. p.W56X was inherited from his mother and p.R384X was from his father. Since both alleles contain nonsense mutations, a lack of CYP11B2 activity was speculated to cause his condition. To our knowledge, this is the first Japanese patient in which the molecular basis of aldosterone synthase deficiency type 1 has been clarified. This case also indicates that spot urinary steroid analysis is useful for diagnosis.

Molecular analysis of the GATA3 gene in five Japanese patients with HDR syndrome.

GATA3 is a member of the GATA family of transcription factors. Heterozygous GATA3 abnormalities are associated with hypoparathyroidism, sensorineural deafness, and renal abnormality (HDR syndrome). However, this triad of symptoms does not occur in all HDR patients and other clinical features may be present in some cases. We report the clinical phenotypes and the molecular analysis of GATA3 in five Japanese HDR patients, including two familial cases. All five patients had hypoparathyroidism and sensorineural deafness, however renal abnormalities were absent in four patients. In addition, two patients with different mutations of GATA3 had female genital tract abnormalities. Sequence analysis of GATA3 demonstrated three novel (R262G, c1063delC and C318) and two reported mutations (c.432insG and c.1051-1G>T). Transient transfection assay using the GATA3 activating reporter system revealed that the transactivating activity of the R262G, c.1063delC, C318S and c.432insG mutants were markedly decreased, indicating that all four mutations are loss-of-function. In conclusion, this study reiterates the clinical variability in HDR syndrome and identifies three novel mutations of GATA3.

The Boy:Girl Ratio of Children Diagnosed with Growth Hormone Deficiency-Induced Short Stature Is Associated with the Boy:Girl Ratio of Children Visiting Short Stature Clinics.

BACKGROUND: About twice as many boys as girls undergo growth hormone (GH) therapy in GH deficiency (GHD). However, this sex difference may not correctly reflect a real incidence. OBJECTIVES: We analyzed the evidence of a selection bias whereby more boys seek treatment at short stature clinics. SUBJECTS AND METHODS: The present study included 3,902 children who visited 17 short stature clinics with a height SD score of -2 SD or less. The percentage of children who underwent the GH stimulation test was compared between boys and girls, as was the percentage of children ultimately diagnosed with GHD. RESULTS: The children comprised 2,390 boys (61.3%) and 1,512 girls (38.7%), with a boy:girl ratio of 1.58:1. The percentage of children who underwent the GH stimulation test did not differ between boys (45.7%) and girls (49.8%). Among the children who underwent the GH stimulation test, the percentage diagnosed with GHD did not differ significantly between boys (22.0%) and girls (20.1%). The boy:girl ratio of children diagnosed with GHD was 1.59:1. CONCLUSIONS: The boy:girl ratio of children with short stature (1.58:1) did not differ significantly from that of children diagnosed with GHD (1.59:1). These results indicate that the predominance of boys in GHD does not reflect a real incidence, but rather a selection bias whereby a higher proportion of boys with short stature seek treatment at clinics. This difference arises because parents are more concerned about boys' height, and because boys reach adult height at an older age.

Targeted Next-Generation Sequencing for Congenital Hypothyroidism With Positive Neonatal TSH Screening.

PURPOSE: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder; however, its molecular etiology remains poorly understood. METHODS: We performed genetic analysis of 24 causative genes using next-generation sequencing in 167 CH cases, comprising 57 dyshormonogenesis (DH), 32 dysgenesis (TD) and 78 undiagnosed. The pathogenicity of variants was assessed by the American College of Medical Genetics guidelines, inheritance pattern, and published evidence. Furthermore, we compared the oligogenic groups and monogenic groups to examine the correlation between variant dosage and severity. RESULTS: We identified variants in 66.5% cases (111/167) and 15 genes, DUOX2, TSHR, PAX8, TG, TPO, DUOXA2, JAG1, GLIS3, DUOX1, IYD, SLC26A4, SLC5A5, SECISBP2, DIO1, and DIO3. Biallelic variants were identified in 12.6% (21/167), oligogenic in 18.0% (30/167), and monogenic in 35.9% (60/167); however, 68.5% of variants were classified as variant of unknown significance (VUS). Further examinations showed that 3 out of 32 cases with TD (9.4%) had pathogenic variants (2 of TSHR and 1 of TPO), and 8 out of 57 cases with DH (14.0%) (7 of DUOX2, 1 of TG) had pathogenic variants. In addition, TSH levels at the first visit were significantly higher in the oligogenic group than in the monogenic group. CONCLUSIONS: The detection rate of pathogenic variants in Japanese CH was similar to that previously reported. Moreover, oligogenic cases were likely to be more severe than monogenic cases, suggesting that CH may exhibit a gene dosage effect. Further analysis of VUS pathogenicity is required to clarify the molecular basis of CH.

OTX2 loss of function mutation causes anophthalmia and combined pituitary hormone deficiency with a small anterior and ectopic posterior pituitary.

CONTEXT: Orthodenticle homeobox 2 (OTX2) is a transcription factor necessary for ocular and forebrain development. In humans, heterozygous mutations of OTX2 cause severe ocular malformations. However, whether mutations of OTX2 cause pituitary structural abnormalities or combined pituitary hormone deficiency (CPHD) has not been clarified. OBJECTIVES: We surveyed the functional consequences of a novel OTX2 mutation that was detected in a patient with anophthalmia and CPHD. PATIENT: We examined a Japanese patient with growth disturbance, anophthalamia, and severe developmental delay. He showed deficiencies in GH, TSH, LH, FSH, and ACTH. Brain magnetic resonance imaging revealed a small anterior pituitary gland, invisible stalk, ectopic posterior lobe, and Chiari malformation. RESULTS: Sequence analysis of OTX2 demonstrated a heterozygous two bases insertion [S136fsX178 (c.576-577insCT)] in exon 3. The mutant Otx2 protein localized to the nucleus, but did not activate the promoter of the HESX1 and POU1F1 gene, indicating a loss of function mutation. No dominant negative effect in the presence of wild-type Otx2 was observed. CONCLUSION: This case indicates that the OTX2 mutation is a cause of CPHD. Further study of more patients with OTX2 defects is necessary to clarify the clinical phenotypes and endocrine defects caused by OTX2 mutations.

Case study of a 15-year-old boy with McCune-Albright syndrome combined with pituitary gigantism: effect of octreotide-long acting release (LAR) and cabergoline therapy.

The use of octreotide-LAR and cabergoline therapy has shown great promise in adults with acromegaly; however, the experience in pediatric patients has rarely been reported. We described a clinical course of a 15-year-old boy of McCune-Albright syndrome (MAS) with pituitary gigantism. At the age of 8 years, a growth hormone (GH) and prolactin (PRL) producing pituitary adenoma was diagnosed at our hospital. He also had multiple fibrous dysplasia, so that he was diagnosed as having MAS. The tumor was partially resected, and GNAS1 gene mutation (R201C) was identified in affected tissues. We introduced octreotide to suppress GH secretion (100 mug 2/day s.c). During therapy with octreotide, IGF-1 and GH levels could not be suppressed and the patient frequently complained of nausea from octreotide treatment. Therefore, the therapy was changed to monthly injections of octreotide-LAR at the age of 12.3 years and was partially effective. However, as defect of left visual field worsened due to progressive left optic canal stenosis, he underwent second neurological decompression of the left optic nerve at 13.4 years of age. After surgery, in addition to octreotide-LAR, cabergoline (0.25 mg twice a month) was started. This regimen normalized serum levels of GH and IGF-1; however, he showed impaired glucose tolerance and gallstones at 15.7 years of age. Therefore, the dose of octreotide-LAR was reduced to 10 mg and the dose of cabergoline increased. This case demonstrated the difficulty of treating pituitary gigantism due to MAS. The use of octreotide-LAR and cabergoline should be considered even in pediatric patients; however, adverse events due to octreotide-LAR must be carefully examined.

Two heterozygous mutations of the AMH gene in a Japanese patient with persistent Müllerian duct syndrome.

Persistent Müllerian duct syndrome (PMDS) is an autosomal recessive disorder of sex development (DSD) characterized by the presence of Müllerian duct derivatives in 46, XY phenotypic males. To date, more than 50 different mutations of the anti-Müllerian hormone gene (AMH) have been reported. Here, we report two novel mutations of AMH in a Japanese patient with PMDS. A 1-year-old male presented with bilateral cryptorchidism and normal male external genitalia. A laparoscopic surgery revealed a uterus and fallopian tubes. Serum AMH was very low. The patient's elder brother was also diagnosed as having PMDS at another hospital. Genetic analysis of AMH showed two novel mutations of p.N486T and p.V527L. Given that these two amino acids are well conserved among different species of AMH, the substitution of two amino acids might affect the normal function of AMH. In conclusion, PMDS should be included in differential diagnoses of cryptorchidism.

Neonatal screening and a new cause of congenital central hypothyroidism.

Congenital central hypothyroidism (C-CH) is a rare disease in which thyroid hormone deficiency is caused by insufficient thyrotropin (TSH) stimulation of a normally-located thyroid gland. Most patients with C-CH have low free thyroxine levels and inappropriately low or normal TSH levels, although a few have slightly elevated TSH levels. Autosomal recessive TSH deficiency and thyrotropin-releasing hormone receptor-inactivating mutations are known to be genetic causes of C-CH presenting in the absence of other syndromes. Recently, deficiency of the immunoglobulin superfamily member 1 (IGSF1) has also been demonstrated to cause C-CH. IGSF1 is a plasma membrane glycoprotein highly expressed in the pituitary. Its physiological role in humans remains unknown. IGSF1 deficiency causes TSH deficiency, leading to hypothyroidism. In addition, approximately 60% of patients also suffer a prolactin deficiency. Moreover, macroorchidism and delayed puberty are characteristic features. Thus, although the precise pathophysiology of IGSF1 deficiency is not established, IGSF1 is considered to be a new factor controlling growth and puberty in children.

Results from 28 years of newborn screening for congenital adrenal hyperplasia in sapporo.

The primary goal of newborn mass screening (MS) for congenital adrenal hyperplasia (CAH) is the prevention of life-threatening salt-wasting crisis in the most severe forms of CAH, and MS for CAH has been implemented in several countries. We summarize here our experience and results from newborn CAH MS from 1982 to 2010 in Sapporo City. During these 28 yr, the level of 17-hydroxyprogesterone (17-OHP) was determined in MS of samples from 498,147 newborns. During this period, 26 individuals (19 females and 7 males) with 21-hydroxylase deficiency (21-OHD) were detected. Of the 26 CAH, 20 were classified as having the salt-wasting (SW) form, 4 were classified as having the simple virilizing (SV) form, and 2 were classified as having the noncalssic (NC) form. Therefore, the frequency of the classical type of CAH was 1 in 20,756. In order to improve the effectiveness, we employed high-performance liquid chromatography (HPLC) as a second tier test from 2000. During this period, among the recalled babies, 75.4% were born prior to the 37th wk of gestation age, and the recall rate was 5.38% for premature neonates and 0.06% for mature neonates. MS for CAH in Sapporo is effective for the identification of the SW and SV forms of 21-OHD. However, the recall rate of premature babies is still high after the introduction of HPLC as a second tier test.

Molecular and Clinical Findings in Patients with LHX4 and OTX2 Mutations.

The pituitary gland produces hormones that play important roles in both the development and homeostasis of the body. Ontogeny of the anterior and posterior pituitary is orchestrated by inputs from neighboring tissues, cellular signaling molecules and transcription factors. Disruption of expression or function of these factors has been implicated in the etiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1F1, LHX3, LHX4, OTX2, SOX2, SOX3 and GLI2. This review focuses on summarizing most recent mutations in LHX4 and OTX2 responsible for pituitary hormone deficiency. In both genetic defects of LHX4 and OTX2, there is high variability in clinical manifestations even in the same family. In addition, there is no clear phenotype-genotype correlation. These findings indicate that the other genetic and/or environmental factors influence the phenotype. In addition, the variability might reflect a plasticity during pituitary development and maintenance. Over the past two decades, a genetic basis for pituitary hormone deficiency and the mechanism of pituitary development have been clarified. It should be kept in mind that this review is not comprehensive, and defects of other transcriptional factors have been described in patients with CPHD. Furthermore, the causes in many patients with CPHD have not yet been determined. Therefore, continuing efforts for the clarification of the etiology are necessary.

Development of endometrial carcinoma in a patient with leprechaunism (donohue syndrome).

Leprechaunism is a rare autosomal recessive disease that is characterized by severe insulin resistance. This disease is caused by a defective insulin receptor and features abnormal glucose metabolism and retarded intrauterine and postnatal growth. However, there are few reports on the long-term course of leprechaunism. We reported the long-term clinical course and rh-IGF-1 treatment in a patient with leprechaunism. During follow-up her diabetes gradually deteriorated despite of treatment of rh-IGF-1. Furthermore, she developed endometrioid adenocarcinoma at the age of 24 yr. The development of endometrial disease must be carefully followed up in this disease.

Loss-of-function and gain-of-function mutations of calcium-sensing receptor: functional analysis and the effect of allosteric modulators NPS R-568 and NPS 2143.

OBJECTIVE: Activating mutations in the calcium-sensing receptor (CASR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CASR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CASR mutants was studied in vitro. METHODS: DNA sequence analysis of the CASR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CASR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CASR. RESULTS: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The activating and inactivating mutations caused leftward and rightward shifts, respectively, in the dose-response curves of the signaling pathway. NPS R-568 rescued the signal transduction capacity of 2 inactivating mutants without increasing cell surface expression levels. NPS 2143 suppressed the enhanced activity of the activating mutants without altering cell surface expression levels, although A843E, which is a constitutively active mutant, was suppressed to a lesser degree. CONCLUSIONS: We have identified 4 novel mutations of CASR. Moreover, our results indicate that allosteric modulators can restore the activity of the loss- and gain-of-function mutant CASRs, identified in this study.

A rare association between Rathke's cyst and hypophysitis in a patient with delayed sex development and growth failure.

We report an 18-year-old Japanese male with a lack of secondary sex characterization and growth failure caused by a rare association between Rathke's cyst and hypophysitis. He was referred to us because of delayed secondary sex characterization. Endocrinological examination showed panhypopituitarism, and the replacement of hydrocortisone, levothyroxine, and desmopressin acetate (DDAVP) was initiated. Brain magnetic resonance imaging (MRI) showed a suprasellar region and a swollen pituitary stalk. The mass was partially resected using the transsphenoidal approach. The pathological diagnosis was hypophysitis and Rathke's cyst. Follow-up MRI performed 1 year after surgery showed that the size of sellar region had not changed. After surgery, in addition to pre-operative hormonal replacement, growth hormone and testosterone were initiated. Two years later, the size of sellar region remains unchanged. In conclusion, while an association between Rathke's cyst and hypophysitis is rare, we suggest that this condition should be included in differential diagnosis of the sellar region, even in adolescents.

Polyarthritis caused by methimazole in two Japanese patients with graves' disease.

In many countries, methimazole (MMI) therapy is the first-line treatment in children with Graves' disease (GD). The rate of side effects of antithyroid drugs (ATDs) in children has been reported to range between 6% and 35%. Of these side effects, polyarthritis is uncommon but serious, and can also develop as a part of the antineutrophil cytoplasmic antibody-associated vasculitis that is induced by ATDs. Here, we describe two GD girl patients aged 15 years and 11 years who developed polyarthritis. The onset of polyarthritis in these patients was 24 days and 28 days after the initiation of MMI therapy, respectively. MMI was suspected of causing the polyarthritis in the two patients and was withdrawn. The symptoms of polyarthritis disappeared rapidly following cessation of treatment. Subsequently, one patient was treated with 131I therapy and the other patient was subjected to thyroidectomy. Although it rarely occurs in pediatric GD patients, severe polyarthritis is a serious side effect of MMI and is an indication for prompt cessation of treatment.

Three novel IGSF1 mutations in four Japanese patients with X-linked congenital central hypothyroidism.

CONTEXT: Congenital central hypothyroidism (C-CH) is a rare disease. We investigated the molecular basis of unexplained C-CH in 4 Japanese boys. PATIENTS AND METHODS: C-CH was diagnosed by low free T4 and/or T3 and low basal TSH concentrations. We used whole-exome sequencing of one patient with C-CH to identify potential disease-causing mutations. Thereafter, PCR direct sequencing was performed to Identify genetic defects underlying C-CH in 3 more patients. We then assessed the effects of mutations identified in the Ig superfamily, member 1 (IGSF1), gene on protein expression and membrane trafficking. RESULTS: All patients had congenital hypothyroidism, and 2 had definitive prolactin deficiency. Two patients were detected by neonatal screening. The other patients were diagnosed by short stature and failure to thrive. We identified a novel nonsense variant in IGSF1 by whole-exome sequencing in patient 1, which was confirmed by PCR direct sequencing (p.R1189X). PCR direct sequencing identified the identical nonsense mutation in patient 2. Patients 3 and 4 harbored distinct missense (p.V1082E) or nonsense (p.Q645X) mutations in IGSF1. The mothers of patients 1, 3, and 4 were heterozygous for these mutations. The R1189X mutant, which lacks the transmembrane domain, failed to traffic to the plasma membrane. V1082E could be observed at the cell surface, but at greatly diminished levels relative to the wild-type form of the protein. The severely truncated Q645X mutant could not be detected by Western blot. CONCLUSION: Our findings provide additional genetic evidence that loss-of-function mutations in IGSF1 cause an X-linked form of C-CH and variable prolactin deficiency.