Screening of SLC26A4 (PDS) gene in Pendred's syndrome: a large spectrum of mutations in France and phenotypic heterogeneity.

Sensorineural hearing defect and goiter are common features of Pendred's syndrome. The clinical diagnosis of Pendred's syndrome remains difficult because of the lack of sensitivity and specificity of the thyroid signs. The identification of PDS as the causative gene allowed molecular screening and enabled a re-evaluation of the syndrome to identify potential diagnostic characteristics. This report presents the clinical and genotypic findings of 30 French families, for whom a diagnosis of Pendred's syndrome had been made. Twenty-seven families had at least one mutated allele. Twenty-eight different mutations were identified, 11 of which had never been previously reported. The main clinical characteristics were: early hearing loss, fluctuation in terms of during deafness evolution, and the presence of an enlarged vestibular aqueduct.

Townes-Brocks syndrome: detection of a SALL1 mutation hot spot and evidence for a position effect in one patient.

Townes-Brocks syndrome (TBS) is an autosomal dominant developmental disorder characterized by anal and thumb malformations and by ear anomalies that can affect the three compartments and usually lead to hearing loss. The gene underlying TBS, SALL1, is a human homolog of the Drosophila spalt gene which encodes a transcription factor. A search for SALL1 mutations undertaken in 11 unrelated affected individuals (five familial and six sporadic cases) led to the detection of mutations in nine of them. One nonsense and six different novel frameshift mutations, all located in the second exon, were identified. Together with the previously reported mutations [Kohlhase et al., 1999], they establish that TBS results from haploinsufficiency. The finding of de novo mutations in the sporadic cases is consistent with the proposed complete penetrance of the disease. Moreover, the occurrence of the same 826C>T transition in a CG dimer, in three sporadic cases from the present series and three sporadic cases from the other series [Kohlhase et al., 1999] (i.e., six of the eight mutations identified in sporadic cases), reveals the existence of a mutation hotspot. Six different SALL1 polymorphisms were identified in the course of the present study, three of which are clustered in a particular region of the gene that encodes a stretch of serine residues. Finally, the chromosome 16 breakpoint of a t(5;16)(p15.3;q12.1) translocation carried by a TBS-affected individual was mapped at least 180 kb telomeric to SALL1, thus indicating that a position effect underlies the disease in this individual.

Two cases of Townes-Brocks syndrome with previously undescribed anomalies.

Townes-Brocks syndrome is characterized by the association of anorectal, radial ray and outer ear malformations and deafness. We describe two patients affected with several typical clinical signs of Townes-Brocks syndrome in addition to growth and puberty delays and vertebral anomalies not previously reported.

Three-year data from a comparative study with recombinant human growth hormone in the treatment of short stature in young children with intrauterine growth retardation.

Growth acceleration and bone maturation were studied for 3 y in 69 children with severe short stature and a history of intrauterine growth retardation (IUGR), to determine the effect of treatment with recombinant human growth hormone (r-hGH). The patients were enrolled in an open, multicentre trial and were randomly allocated to either the treated group (Group 1) or the control group (Group 2). The children in Group 1 were treated daily with 0.2 IU/kg/body weight (0.067 mg/kg) s.c., during 3 y and the children in Group 2 started the study with a 1-y observation period followed by a 3-y treatment period. At birth, their mean weight standard deviation score (SDS) was -2.5 and their mean length SDS -3.5. At baseline, the patients were prepubertal, non-GH deficient, with no known dysmorphic features. Mean age was 4.5 y, bone age was 3.3 y, height SDS was -3.4, height velocity (HV) SDS was -1.6, and body mass index SDS was -1.4. After 1 y of treatment, linear HV in Group 1 increased in comparison with the pre-treatment period (from 5.7 +/- 2.0 to 10.1 +/- 1.7 cm/y; p < 0.001) and with the first year of observation in Group 2 (p < 0.001). Increased HV was sustained during the second and third year of treatment and was significantly higher than at baseline. A similar growth pattern was seen during the 3 y of GH treatment in Group 2. Mean height SDS for chronological age increased by 2.0 +/- 0.7 in the two groups after 3 y of treatment. HV after 1 y of treatment was negatively correlated with growth velocity at baseline. Bone age remained retarded but increased with a mean of almost 4 y after 3 y of treatment in both groups. Even at a dose that is three times the replacement dose treatment with r-hGH was well tolerated. From these results, we conclude that r-hGH treatment over 3 y can induce sustained catch-up growth in young children with severe short stature and a history of IUGR. Long-term studies are needed to assess ultimate effects on final height.

[Results of national evaluation tests (primary schools) of CE2 and 6th classes of 73 students with congenital hypothyroidism screened at birth]. / Résultats des tests nationaux d'évaluation (scolarité primaire) de CE2 et de 6e chez 73 élèves hypothyroïdiens congénitaux dépistés à la naissance.

UNLABELLED: Assessment of school performance provides an excellent index of adequacy of therapy of congenital hypothyroidism. PATIENTS AND METHODS: Scholarly achievement of 73 children with congenital hypothyroidism screened and followed-up in our clinic was evaluated by comparison to national tests calculated from a wide random sample. The 73 children were divided into three groups: 31 patients belonging to the CE2 class (third primary school class) (group 1); 13 patients belonging to the 6th class (first secondary school class) having already passed CE2 tests (group 2); and 16 patients also belonging to the 6th class but not having passed CE2 tests (group 3). RESULTS: The youngest patients showed better results in French than national means, with similar results in mathematics. The intermediate aged patients showed comparable scores to the national mean in both tests. The oldest patients showed significantly less achievement in mathematics, and somewhat (but not markedly) less achievement in French. Etiology of hypothyroidism did not seem to be a prognostic factor, with the athyreotic patients having better results in CE2 than ectopic ones. Age at onset of treatment and the duration of treatment before normalization of thyroid stimulating hormone (TSH) seemed to represent the main prognostic factors as does compliance to treatment, which depends on socioeconomic levels of families. CONCLUSION: Encouraging results are now obtained in those patients who have been screened in the neonatal period and treated very early. Compliance to the treatment remains an important factor of prognosis.

[Psychosocial and sexual outcome in women with Turner syndrome]. / Enquête sur l'avenir socio-psycho-affectif des femmes atteintes de syndrome de Turner.

Results of an inquiry in adults patients (18-53 years of age) coming from three centers (1 in Rouen & 2 in Paris), 213 questionnaires were sent. 105 answers were received. Scholar achievement: only secondary cycle in 6%, Secondary cycle + professional course in 22% and tertiary cycle in 44%. No scholar ship in 2%, 26% were still ongoing studies. Professions: 18% are unemployed (24%), Secretary jobs: 10%, Health professions (altogether): 18%, Teachers: 8%, Clerks: 7%, Executive jobs: 8% and Miscellaneous jobs: 5%. 3% have an handicapped status. The small height was a career obstacle in 29%. Affective life. Age of first sexual intercourse was 19-22 years. 17 are or were married and 15% are living in couple. But 58% have not any sexual life whatsoever. These women are divided on the ways to cure the sterility. Few among the oldest have attempted adoption or medically assisted procreation, with each time low rate of success. 26% have psychological disturbances which were serious in 6% mainly due to depression.

Modifications of growth hormone secretion during female puberty.

Growth hormone levels rise steadily through normal puberty, in parallel with the pubertal stages but decline rapidly at the end of puberty (stage V). The general evolution of the secretory profile of GH is parallel to the growth velocity curve. The frequency of GH pulses remains unchanged; however, their amplitude, mean integrated concentrations, area under the curve, and urinary growth hormone are elevated at midpuberty. The main action of GH is to ensure, together with sex steroids, the pubertal growth spurt. However, the role of pubertal GH is not confined to inducing the pubertal growth spurt. It also participates, together with sex steroids, in the acquisition of adult bone mineral density.

A thyroxine dosage of 8 micrograms/kg per day is appropriate for the initial treatment of the majority of infants with congenital hypothyroidism.

UNLABELLED: The adequate L-thyroxine dosage for the initial treatment of infants with congenital hypothyroidism is a subject of controversy. Some recommend higher dosages (> 10 micrograms/kg/day) to ensure adequate levels, while others advocate lower dosages to permit normalisation of thyroid status. The aim of this study was to evaluate the results of a treatment strategy using an initial dosage of 7.5-8.0 micrograms/kg per day, TSH measurements being taken at 15 and 30 days of treatment. Fifty one newborns infants with primary congenital hypothyroidism detected by neonatal screening were treated with the same therapeutic strategy. A mean L-thyroxine dosage of 7.9 micrograms/kg per day at the onset of treatment and 6.6 micrograms/kg/d at 2 months, normalised the FT4 and FT3 levels at 15 days in 100% and TSH levels at 2 months in 90% of cases. Many patients showed elevated levels of FT4 and a systematic higher initial dosage could expose many infants to a dangerous hyperthyroidism. Patients with abnormal TSH levels at 2 months already had higher TSH levels in the first 8 weeks of life and, despite higher L-thyroxine dosage, also exhibited lower FT4 and FT3 levels. These patients who needed an early increase in dosage had already shown a more profound ante and neonatal hypothyroidism. This subgroup of patients require a higher dosage of thyroxine and early assessment of FT4, FT3 and TSH levels are required for optimum dosage choice. CONCLUSION: Even though a subgroup of patients may require a higher dosage of L-thyroxine, an initial dosage of 7.5-8.0 micrograms/kg per day, with an early assessment of FT4, FT3, and TSH levels, is adequate for the treatment of the majority of infants with congenital hypothyroidism.

Four families with loss of function mutations of the thyrotropin receptor.

We observed four families with loss of function mutations of the TSH receptor gene. One patient had a homozygous Pro162 Ala substitution. The three other were compound heterozygotes: 1) Gln324-->Stop and Asp410 Asn2), Cys41 Ser and Phe525 Leu, 3) Cys390 Trp and Trp546-->Stop. In all patients, the plasma TSH concentration was increased, whereas T3 and T4 concentrations were normal. The TSH levels were normal in the heterozygous parents. These results confirmed the recessive character of TSH receptor defects. Expression of the various mutated receptors in transfected COS-7 cells demonstrated the impairment of their function. We studied the expression of the receptors on the cell surface by immunofluorescence, their ability to bind hormone, and their capacity to activate adenylate cyclase. Some mutations allowed us to identify sites that are especially important for receptor function. The substitution Cys390 Trp abolished high affinity hormone binding. Receptor mutated at Asp410 Asn bound the hormone normally, but failed to activate adenylate cyclase. This result underscores the role of this acidic extracellular residue, close to the first transmembrane segment, in signal transmission. The Phe525 Leu substitution also markedly impaired adenylate cyclase activation, underlining the importance of the second intracellular loop in receptor signaling.

[Alpha fetoprotein in congenital hypothyroidism before and during treatment]. / L'alphafoetoprotéine au cours de l'hypothyroïdie congénitale avant et sous traitement.

BACKGROUND: Screening for congenital hypothyroidism (CH) is an opportunity to investigate maturation by measuring alphafetoprotein (AFP). PATIENTS AND METHODS: Blood AFP was measured in 73 full-term infants (controls), 22 infants with permanent CH and 19 with a transient form (FT) of hypothyroidism. It was also measured in mothers of the two groups with hypothyroidism. AFP was measured by RIA and its value was compared to those of FT4, TSH and bilirubin. RESULTS: Blood AFP was higher in patients with CH with significant differences between patients who had jaundice or not, but AFP was not significantly different in FT patients and controls. Initial values of AFP in both CH and FT population was correlated to the levels of AFP in their mothers. Under treatment with 7.5 micrograms/kg/day of l-T4, AFP levels remained increased at T15, then gradually normalized at T30-T60. The log of AFP was correlated to TSH levels between T15 and T60 but was not correlated to FT4 levels. Preliminary results at one year of age show that IQ seems better in infants with early normalization of AFP. CONCLUSIONS: Prolonged follow-up is necessary to assess the possibility that initial kinetics of AFP under therapy have a prognostic value for estimating the quality of outcome.

Clinical and anatomical spectrum in XX sex reversed patients. Relationship to the presence of Y specific DNA-sequences.

OBJECTIVE: Testicular differentiation can occur in the absence of the Y chromosome giving XX sex-reversed males. Although Y chromosomal sequences can be detected in the majority of male subjects with a 46,XX karyotype, several studies have shown that approximately 10% of patients lack Y material including the SRY gene. The aim of this study was to see if the classification of XX sex-reversed individuals into three groups, Y-DNA-positive phenotypically normal XX males, Y-DNA-negative XX males with genital ambiguities and Y-DNA-negative true hermaphrodites can be applied to our cases. DESIGN: Endocrinological and genetic studies were conducted in 20 XX sex-reversed patients. PATIENTS: Twenty patients with various phenotypes were studied. They were between 20 days and 35 years old. Ten presented ambiguous external genitalia (Prader's stages II to IV). After laparotomy or gonadal biopsy, the diagnosis was 46,XX true hermaphroditism in five, and XX male in 15. MEASUREMENTS: Blood samples were obtained from all patients for hormonal and molecular studies. Basal levels of testosterone, oestradiol and pituitary gonadotrophins were measured by RIA. In addition, two stimulation tests were performed: gonadotrophin stimulation with GnRH and testicular stimulation with hCG. Several Y-specific DNA sequences of the short arm of the Y chromosome were analysed by Southern blot and polymerase chain reaction methods. RESULTS: In this study, three categories of XX sex-reversed individuals were observed: phenotypically normal males with or without gynaecomastia, males with genital ambiguities, and true hermaphrodites. Endocrinological data were similar in XX males and in true hermaphrodites. Testosterone levels exhibited normal (n = 9) or decreased (n = 11) values. The hCG response was low. FSH and LH were elevated in 13 patients. Molecular analysis in ten patients showed varying amounts of Y material including the Y boundary and SRY. Ten patients with various phenotypes lacked Y chromosomal DNA. There was no relation between Leydig cell function (as indicated by testosterone levels before or after hCG stimulation) and the presence of Y chromosome material. CONCLUSION: Although the presence of Y-specific DNA generally results in a more masculinized phenotype, exceptions do occur. In the Y-DNA-negative group, complete or incomplete masculinization in the absence of SRY suggests a mutation of one or more downstream non-Y, testis-determining genes.

[Management of female precocious puberty]. / Conduite à tenir devant une précocité pubertaire féminine.

Occurrence of any pubertal sign before eight years of age defines premature sexual development but does not always mean precocious puberty (PP); one should distinguish borderline physiological situations which need only a follow-up and frankly pathological situations which need very precise investigations and suitable treatment. The first situations are premature thelarche, pubarche and menarche in which the height and bone maturation, pelvic ultrasonography (US) are normal for age, avoiding hormonal investigations. Conversely in the second situation, the bone age is more advanced than the height age and the pelvic US displays ovarian activity and uterine development. The next step is the characterization of the level of the mechanism of puberty: hypothalamohypophysal or ovarian: in the first case gonadotropin levels are elevated after GnRH infusion, in the second case, depressed. The aetiological diagnosis are in true PP: brain tumors malformations or hamartoma even if negative idiopathic. At ovarian level: ovarian tumors or McCune Albright syndrome or recurrent cysts. The first etiology leads to use GnRH analog in the second the treatment is more delicate.

Molecular prenatal diagnosis of partial androgen insensitivity syndrome based on the Hind III polymorphism of the androgen receptor gene.

OBJECTIVE: Partial androgen insensitivity syndromes are the cause of genital ambiguity that is at times quite severe; there is, therefore, a high demand for prenatal diagnosis in families already afflicted with this syndrome. When the mutation has not been identified, the diagnosis can be made by the study of the polymorphisms of the androgen receptor gene. To perform molecular prenatal diagnosis in a family with partial androgen insensitivity syndrome, we studied the Hind III polymorphism of the androgen receptor gene on the trophoblastic DNA. The use of this restriction fragment length polymorphism tracked maternal X chromosome segregation and established prenatal diagnosis although the mutation had not yet been identified in this family. FAMILY: The mother had been previously described as heterozygous for the Hind III polymorphism and chromosomal segregation analysis showed that the affected allele was associated with the 6.7-kb Hind III fragment. MEASUREMENTS: Hind III RFLP with an androgen receptor gene cDNA probe was realized on the trophoblastic DNA, along with measurement of androgen binding activity on the trophoblastic cells. RESULTS: We detected the presence of the 6.7-kb fragment in the DNA of the trophoblastic cells suggesting the fetus was affected. Partial androgen insensitivity syndrome was confirmed by a considerable decrease in androgen binding activity on the trophoblastic cells and by sonography of the fetus. After a therapeutic abortion requested by the parents, the diagnosis was confirmed by clinical examination of the fetus, biochemical analyses of the fetal androgen receptor, and molecular studies of the fetal DNA. CONCLUSIONS: When the mutation of the androgen receptor gene has not been identified, Hind III polymorphism of the trophoblastic DNA is useful in the prenatal diagnosis of androgen insensitivity syndrome in high-risk families.

[Study of sex determination gene (SRY) in 46,XY gonadal dysgenesis]. / Etude du gène du déterminisme du sexe (SRY) dans les dysgénésies gonadiques 46,XY.

During mammalian embryogenesis, the presence of the SRY gene determines the bipotential gonad to develop as a testis. 46,XY sex reversal has been described in man. It is associated with an essentially female phenotype and a streak gonad. In a collaborative study, we analysed 36 patients with a 46,XY sex reversal. The testis determining region of the Y chromosome was analysed by Southern blotting and by DGGE analysis of the SRY open reading frame (orf). We found a total of 7 mutations in the testis determining region including the SRY gene. This brings to 19 the total number of mutations in SRY associated with sex reversal. No relationship was found between the SRY status and the presence or absence of gonadoblastoma. However, a correlation was observed between the SRY genotype and the histology of the gonad. A mutant in SRY is associated with a completely dysgenetic gonad. The presence of immature testicular tubules is usually observed when SRY is normal. These latter results suggest the existence of as yet unidentified testis determining genes.

Growth of short normal children in puberty treated for 3 years with growth hormone alone or in association with gonadotropin-releasing hormone agonist.

GH, 0.1 IU/kg/day 6 days/week, was given to 30 early pubertal short patients for 3 years. There were 16 males, aged 14.4 +/- 0.8 years, and 14 females, aged 12.2 +/- 1.2 years, at pubertal stage 2 or 3 with slow growth (4.2 +/- 1.2 cm/year) and no detected GH insufficiency or other cause for short stature. They were randomized in 2 groups: group A with GH alone, and group B with GH and a gonadotropin-releasing hormone agonist during the first 2 years. 28 of the 30 patients completed 3 years of treatment. The annual growth rate increased during the 1st year in both groups and sexes, the increase being significant (p < 0.01) in group A only. Patients of group A kept an improved growth velocity in the 2nd year, then returned to pretreatment growth rate in the 3rd year, while completing their sexual development and bone maturation. Their height, expressed as standard deviation score (SDS) for bone age, improved in the first 2 years, but decreased thereafter. Group B patients returned to pretreatment growth velocity in the 2nd year, and had no significant improvement in growth rate in the 3rd year with GH alone. Their bone maturation, slow when on the GnRH agonist, accelerated when sexual development resumed. At the end of the 3 years, height, expressed as SDS for age, improved in group A from -2.5 +/- 0.6 to -1.5 +/- 0.4 in males (p < 0.05) and from -2.8 +/- 0.5 to -2.1 +/- 0.9 in females (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal and molecular genetic findings in 46,XX subjects with sexual ambiguity and testicular differentiation.

Ten patients were studied who had sexual ambiguity having in common a 46.XX karyotype and testicular tissue. They were aged from one month to 23 years; some of them were followed through puberty. Eight cases were sporadic and two familial. They were divided into two groups according to finding of surgery and histology: 46, XX males with sexual ambiguity and 46 XX true hermaphrodites (TH). They were no differences in phenotypes (except uterus and ovotestis in TH). The endocrinological data were identical in the two groups: testosterone levels were in the normal range during puberty, then decreased in adulthood. Gonadotrophins were above the normal range at mid-puberty. Gonadal biopsies, regardless of the ovarian part of the ovotestis, were identical in two groups, i.e., normal in the youngest patients, then spermatogonia disappeared afterwards and dysgenesis became obvious. In one case, the ovarian zone of the ovotestis was only detected on serial cuts after gonadectomy. Southern blots displayed the presence of Y specific material in tow cases (PABY-SRY-PO.9). Otherwise, in all other patients, there was the lack of any Y sequences without any differences between the two groups. These data suggests that 46, XX males with sexual ambiguity and 46 XX true hermaphrodites may be alternative expressions of two genetic defects: one, a minimal interchange between Yp and Xp, another, a mutation of an autosomal testis determining factor for the patients without Y detectable material.

Prenatal prediction of androgen insensitivity syndrome using exon 1 polymorphism of the androgen receptor gene.

Exon 1 polymorphism of the androgen receptor (AR) gene is characterized by a (CAG)n(CAA) repeat at position 172 following the translation start codon. The aim of this study was to determine whether AR gene exon 1 polymorphism could be used to perform prenatal diagnosis in high risk families with complete or partial androgen insensitivity syndrome. After enzymatic amplification of a 1 kilobase exon 1 fragment, each DNA was simultaneously digested by MspI and PstI restriction enzymes. After electrophoresis on a 15% electrophoresis on a 15% acrylamide gel or a 6% Nusieve gel, we measured the size of the obtained fragments and determined the number of CAG repeats since a 282 basepair fragment corresponds to 21 CAG. We previously showed that the number of CAG repeats within the AR gene exon 1 in 23 families with complete or partial androgen insensitivity syndrome was 19 +/- 4. By this method, we detected heterozygosity in 50% of the mothers. We present here 2 exclusion prenatal diagnoses using exon 1 polymorphism of the AR gene. Family A presented a boy with a severe form of partial androgen insensitivity syndrome. The mother had 2 uncles with ambiguous genitalia. In family B, the affected child had a complete androgen insensitivity syndrome. In both families, analysis of the AR gene exon 1 polymorphism of the trophoblastic DNA showed the presence of the normal maternal X chromosome. The parents decided to carry on the gestation. In family A, the newborn had normal male external genitalia. In family B, sonography confirmed the presence of normal male external genitalia. These data suggest that exon 1 polymorphism of the AR gene could be prenatally used to predict androgen insensitivity syndrome.

Screening for Y-derived sex determining gene SRY in 40 patients with Turner syndrome.

In Turner patients, the presence of a Y chromosome or derivative Y is correlated with the risk of gonadoblastoma induction. "Marker" chromosomes originating from Y, may not show characteristic fluorescence and then be very difficult to identify by conventional cytogenetic techniques, although they still predispose the patients to gonadal tumors. Using polymerase chain reaction of the gene from the sex-determining region of the Y chromosome, we screened 40 Turner patients (thirty seven 45X and three 45X,46XX) for the presence of Y chromosomal DNA. We were able to identify karyotypically unrecognized Y chromosome material in 1 patient out of the 40 studied. In this patient mild clinical and biological hyperandrogenism was observed. Reliability of our technique was ascertained by the detection of the expected 648 base pairs amplified DNA fragment in all normal male controls as well as in 3 Turner patients with confirmed 45X,46XY mosaicism. Despite the low frequency of unrecognized Y chromosome material (1 case over 40 in our experience), our data suggest that polymerase chain reaction of the gene from the sex-determining region of the Y chromosome is worthy of being performed in Turner patients considering the potential risk of the presence of a Y chromosome.