CLINICAL ANALYSES (UNPUBLISHED DATA)

Patients referred to the Department of Clinical Genetics at Karolinska University Hospital, Stockholm, Sweden, for genetic investigation of gonadal DSD and POI have been considered for this thesis. For these patients, the initial investigation of choice is a conventional karyotype. If an aberration is identified, this is often diagnostic. Further investigations that can be offered include:

 46,XY gonadal DSD: Sequence analysis of SRY, NR5A1, WT-1, SOX9, DHH and MLPA analysis detecting aberrations in NR0B1, SOX9, SRY, WNT4 and NR5A1.

 46,XX gonadal DSD: Sequence analysis of SRY and NR5A1. MLPA analysis can be considered depending on clinical presentation.

 POI: Sequence analysis of BMP15, FSHR and NR5A1. FMR1 analysis in SA.

Some patients have received a molecular diagnosis by routine clinical genetic investigation or in previous research studies. An overview is given in table 8. The remaining patients have been included in the studies in this thesis.

XY gonadal DSD

Three patients with 46,XY GD have been diagnosed with SRY point mutations, and one patient has a diagnostic SRY deletion. One patient has a missense mutation in the WT-1 gene considered causative. Also different forms of chromosomal mosaicism have been found in three patients. For the remaining 14 patients with GD, clinical genetic

investigations have not revealed any causative mutation. Among the three patients with other gonadal DSD forms, one patient with mixed GD has a causative dicentric Y-chromosome rearrangement.

Previous research studies in the group using BAC array and MLPA analysis, have identified a pair of affected siblings carrying NR0B1 duplications [90], detected before the development of the MLPA set now used at the Department of Clinical Genetics.

Also one patient with a causative 9p deletion has been identified [69].

The overall diagnostic rate in 46,XY GD can be estimated to 44% in our material.

Previous reports vary between 20-50% [15].

Table 8. Results of clinical genetic investigations.

Clinical diagnosis Patients studied (n) 46,XY complete GD 14 25 SRY mutation/deletion (4)

WT1 mutation (1)

GD, gonadal dysgenesis; Del, deletion; DSD, disorders of sex development; POI, primary ovarian insufficiency; PA, primary amenorrhea; SA, secondary amenorrhea.

XX gonadal DSD

One patient with 46,XX testicular DSD has been diagnosed as SRY positive, explaining the phenotype. The remaining patient with 46,XX testicular DSD, 46,XX ovotesticular DSD and the patient with one streak gonad and one functioning ovary, have not received a molecular diagnosis.

POI

Four patients with POI had diagnostic aberrant karyotype with either X-chromosome deletion or translocations involving the X-chromosome. One had PA and three had SA.

In three patients, balanced translocations involving autosomes have been identified, but not considered causative. One of these patients has participated in the studies included in this thesis. The remaining two patients have not been available for inclusion.

One patient with SA has been diagnosed with a causative FMR1 premutation. This corresponds to a detection rate of 1/54=2.8% which is expected for unrelated cases (2-5% in previous reports) [24, 44].

In total, 54 patients with POI were included in the study, 23 with PA and 31 with SA.

All patients with POI participating in the studies of this thesis have undergone sequence

analysis of the BMP15, FSHR and NR5A1 genes. No causative mutation has been identified, although three novel changes have been found.

One patient is a heterozygous carrier of a novel silent change in the BMP15 gene (c.393T>C), inherited from the mother. A novel silent change was found in the FSHR gene in another patient in heterozygous form (c.1653C>T), no parental samples were available. The silent changes were not predicted to affect splicing.

In NR5A1 a missense change was identified in one patient with SA, c.771C>G, p.Asp257Glu. The patient is heterozygous, and the mother does not carry the change.

Paternal samples were not available. The amino acid substitution occurs in a nuclear hormone receptor ligand binding domain, but the substitution is, based on prediction tools and the similarity between exchanged amino acids, considered benign.

These patients are included in study II, III and IV of this thesis.

Conclusion

The diagnostic rate is higher for the group with 46,XY gonadal DSD than for 46,XX gonadal DSD and POI. This can be due to differences in mutation frequency of causative genes in 46,XY and 46,XX respectively, reflect that POI is a more

heterogeneous condition, or illustrate that the group with 46,XX gonadal DSD and POI is far less studied than 46,XY.

Through previous research in the group studying 46,XY GD, three patients could receive a molecular diagnosis. This was the rationale to extend the study to also include 46,XX patients and to further analyze the undiagnosed 46,XY patients in the current project.

A karyotype is always necessary for the analysis of patients with gonadal DSD and POI as it is the basis of classification of DSD forms, and can also be diagnostic alone, such as in the case of mosaicism or translocations involving the X chromosome. For patients with SA, FMR1 analysis must be recommended, especially in familial cases. It is a simple and affordable analysis, and there can be undetected younger carriers in the family at risk for POI. In addition, premutation carriers have an increased risk of having children with Fragile-X mental retardation, and must be offered genetic counseling.

Today, there are several known genes in gonadal DSD and POI to investigate in patients. A suitable genetic investigation must be recommended for giving affected patients a molecular diagnosis. Even if not influencing therapy recommendations, a molecular diagnosis will provide the patient with an explanation, which can be some

consolation in a difficult condition. A summary of the clinically relevant genes as of today is given in the concluding remarks.

GENOME WIDE APPROACH TO IDENTIFY NOVEL CANDIDATE