case_images: PMC9592858_01
case_images: Physical examination, clinical signs, and HHAT candidate variant in our 46,XY patient. (A) Microcephaly visible in X-rays. (B) Skeletal survey showed hypoplasia of distal phalanges of both thumbs and bilateral capito-hamate coalition. (C) The anatomopathological analysis of the right and left dysgenetic gonads showed immature seminiferous tubules (ST) with hyperplasia of Leydig cells (LC) (left and magnified image) and a germ-cell (positive for placental alkaline phosphatase (PLAP) staining) tumor in the right gonad (asterisk) (right). (D) Family pedigree (right) and electropherogram (left) showing the proband (II-1, arrow) and parental (I-1 and I-2) genomic DNA with a T>A substitution observed in the 1001 position of HHAT gene (NM_018194). Both parents were carriers.
case_images: fendo-13-957969-g002.jpg
case_images: PMC9592858_01_fendo-13-957969-g002.jpg
case_images: f2
case_images: The skeletal survey at 13.5 years of age revealed microcephaly, with a head circumference of 51.8 cm (SD -2.65) ( Figure 2A ) and hypoplasia of distal phalanges of both thumbs and bilateral capito-hamate coalition ( Figure 2B ). The ophthalmologic evaluation showed an eye fundus with asymmetric papilla (large diameter in right eye), a retinal pigment epithelium atrophy, a temporary flat major excavation in the right eye applied retina, and normal peripheral vision in both eyes. Microscopically, both dysgenetic gonads contained immature seminiferous tubules, 70% of which had germinal cells, and Leydig cell hyperplasia was observed ( Figure 2C , left and magnified images). In the central zone of the right testis parenchyma, a tumor cell proliferation of 5 mm was identified ( Figure 2C , right image). The HHAT (NM_018194) variant c.1001T>A/p.(Met334Lys), located at the end of exon 8, was classified as a variant of uncertain significance (VUS) according to the American College of Medical Genetics and Genomics (ACMG) criteria (PM2 and PP3) ( Figure 2D ).
case_images: Results of the HHAT in vitro minigene functional studies. (A) Results of the HHAT in vitro minigene experiments. COS-7 cells were transfected with wild-type (WT) HHAT, c.1001A mutant HHAT (MT), or both WT/VA HHAT. RNA was extracted, and RT-PCR for HHAT cDNA was performed. The figure shows the PCR products on an agarose gel. (B) Electropherograms of the minigene PCR products. The arrow indicates the position of the nucleotide variant c.1001A. Hom, homozygous state; Het, heterozygous state.
case_images: fendo-13-957969-g003.jpg
case_images: PMC9592858_01_fendo-13-957969-g003.jpg
case_images: f3
case_images: All minigene transfections (WT/WT, WT/MT, and MT/MT) apparently resulted in cDNA amplification products of the same size, close to the length of minigene design including exons 8 to 10, corresponding to 416 bp ( Figure 3A ). The sequence analysis of these products confirmed that all of them conserved the entire sequence of exons 8, 9, and 10 ( Figure 3B ), which means that the variant does not affect splicing and not confirming the in silico prediction of aberrant splicing.
case_images: HHAT p.(Met334Lys) variant in silico studies. (A) Diagram of the HHAT protein isoform NP_060664.2 with its different domains and localization of variant p.(Met334Lys) in membrane-bound O-acyltransferase (MBOAT) functional domain of the protein. The in silico studies using Clustal Omega showed that the mutated Methionine 334 is conserved through vertebrates. (B) 3D image of the HHAT molecule and the ligand palmitoyl-CoA (in pink) indicating the position of Met334 (PBD 7MHY, RCSB PDB). (C) 3D magnified image indicating that the 334 position is very close to the ligand palmitoyl-CoA. (D) HHAT peptide sequence showing the amino acids in the active center (<8 A from the ligand) (in red), which includes Met334 (underlined).
case_images: fendo-13-957969-g004.jpg
case_images: PMC9592858_01_fendo-13-957969-g004.jpg
case_images: f4
case_images: The HHAT variant p.(Met334Lys) is located in the functional MBOAT domain ( Figure 4A ). The conservation across species suggests an important role of this amino acid in the HHAT protein structure, stability, or function ( Figure 4A ). Methionine 334 is one of the nearest (5 A) amino acids to the palmitate donor palmitoyl-CoA ( Figures 4B-D ). Moreover, this single-point variant was predicted to decrease HHAT protein stability, in terms of sequence and protein structure using the same PDB model 7MHY.