Publicación familia trisomía 9p

INFORME CLINICO

Paciente 1 (Fig. 1) La propósita es la tercera hija de padres sanos, no consanguíneos. Tiene siete tíos maternos (no explorados) en dos generaciones anteriores y un hermano con retraso mental (ver árbol genealógico). Nació por cesárea a las 37 semanas de una gestación que cursó con hipocinesia fetal y ruptura prematura de membranas. Al nacimiento pesó 2500g y se le diagnosticó luxación congénita de cadera, sin otras complicaciones neonatales.Presentó retraso psicomotor desde las primeras semanas de vida postnatal, y a los 17 meses la exploración clínica mostró lo siguiente:Peso 7 700k, talla 72cm perímetro cefálico 42cm. Hipotonía muscular generalizada. Cráneo con microbraquicefalia, frontal discretamente prominente. Cara redonda, puente nasal ancho y prominente. Ojos con hipertelorismo, telecanto y epicanto interno. Pabellones auriculares displásicos con implantación baja. Nariz globulosa, columela corta, filtrum corto, y micrognatia. Cuello corto. Tórax con discreta teletelia, no se identificaron soplos cardíacos. Manos con braquimesodactilia, hipoplasia de las uñas y clinodactilia del quinto dedo. Pies pequeños con braquidactilia y uñas hipoplásicas. Pliegues palmares y plantares aberrantes. Genitales femeninos sin alteraciones clínicas aparentes.

Paciente 2 (Fig. 2) hermano de la propósita, escolar masculino, el cual exploramos a los 9 años de edad. Su fenotipo es muy similar al de su hermana, desde su nacimiento presentó retraso psicomotor, actualmente asiste a una escuela de educación especial por presentar disminución del coeficiente intelectual. Nació por cesárea indicada por sufrimiento fetal, después de una gestación de 38 semanas sin otras complicaciones. Peso al nacer 2 800g. A los 24 meses se le practicó hernioplastía de una hernia umbilical y a los 4 años orquidopexia izquiera y orquiectomia derecha. Talla 1.26m, peso 25k, perímetro cefálico 48cm. Microbraquicefalia, frontal estrecho. Cara redonda, puente nasal ancho y prominente, naríz globulosa, columela corta, filtrum corto. Hipertelorismo ocular, fisuras palpebrales antimongoloides. Comisuras bucales hacia abajo, paladar ojival, micrognatia. Pabellones auriculares prominentes, displásicos y con implantación baja. Cuello corto. Tórax sin alteraciones, no se identificaron soplos cardíacos. Manos pequeñas con braquimesodactilia, uñas hipoplásicas y pliegues palmares anormales. Genitales externos masculinos, hipoplasia escrotal y del testículo izquierdo.

IV

I

II

III

Retraso Mental (no explorados)

Portadores de la translocación balanceada ( con estudio citogenético)

Afectados

Fig. 2 Árbol Genealógico Familia trisomía 9p

CLINICAL REPORT

Patient 1 (Fig. 1) The proband is the third product of healthy non-consanguineous 36 years-old father and 27 years-old mother. She has a brother with a similar clinical picture and there is a history of mental retardation (MR) in two previous maternal line generations (Fig. 2). Gestation underwent with diminished fetal movements. Patient was born by cesarean section prompted for early amnion rupture at 37 weeks of pregnancy; her birth weight was 2500g, at this time hip dislocation diagnosis was made.Psychomotor developmental delay was noted too early.In our first clinical evaluation at 17 months-old she presented the following characteristics:weight 7,700K (-2SD), height 72cm (-2SD), cephalic circumference 42cm (-2SD), micro-brachycephaly, round facies, downslating palpebral fissures, ocular hyperthelorism, telecanthus and epicanthic folds, broad and prominent nasal bridge, fleshy tip, short filtrum, downturned corners of the mouth, narrow palatal arch, micrognathia. Low set misshaped ears, short neck. Thorax with slight widespread nipples, no abnormal heart murmurs were found. Small hands and feet with hypoplastic nails, brachymesodactyly of fingers, fifth finger clinodactyly and abnormal palmar creases. Muscle tone was diminished and her genitals show no alteration.

Patient 2 (Fig. ) Is the 9 years old brother with a similar clinical picture of patient 1.He was born by cesarean section for fetal distress at 38 weeks gestation, his birth weight was 2 800g, no other perinatal impairments were detected. He showed early psychomotor developmental delay. At 24 months old he underwent a surgical correction of an umbilical hernia. At 4 years old, after cryptorchidism diagnosis was made, left orchidopexia and right orchiectomy were practiced him. At present evaluation, we found him with the follow clinical abnormalities: weight 25K ( ), height 1.26m ( ), cephalic circumference 48cm ( ). Microcephaly, frontal estrecho, round facies, downslating palpebral fissures, ocular hiperthelorim, telecanthus and epicanthic folds, broad and prominent nasal bridge, fleshy tip, short filtrum, downturned corners of the month, narrow high palatal arch, micrognathia, Low set misshaped prominent ears, short neck. Normal thorax witthout abnormal heart murmurs. Small hands, brachymesodactyly of the fingers, fifth finger clinodactyly, hipoplastic nails, and abnormal palmar creases. External genitals with hypoplastic scrotum and diminished size of the left testicle.

Cont. Table

CINICAL FINDINGS IN TWO MEXICAN CHILDREN WITH TRISOMY 9P

Craneofacial Abnormalities Case 1( girl )

Case 2( boy )

Down turned corner of the mouth + +

Fleshy Tip + +

Misshaped or low set ears + +

Strabism - -

Short Filtrum + +

Hypertelorism +

Microcephaly + +

Brachycephaly + +

Deep-set eyes - +

Narrow Palatal Arch + +

Downslating Palpebral Fisures + +

Micrognatia + +

Skelethal Anomalies + +

Short Stature + -

Diminished Skelethal Maduration - -

Fifth Finger Clinodactyly + +

Brachymesodactyly + +

Hypoplastic Phalanges + +

Hypoplastic Nails + +

Lordosis - -

Scholiosis - -

Short Neck + +

Dislocation of the hip + -

Congenital Heart Defect In course Not explored

Ventricular Septal Defect

Ebstein AnomaliePatent Ductus Arteriosus

Central Nervous System AlterationsMental Deficiency + +Agenesis of Corpus Callosum Ventriculomegalia Cerebellar hypoplasia Choroidal cysts gray matter heterotopiaSeizures - -

Abnormal configuration of Dermal Ridges

+ +

Single palmar Crease - +Single flexor crease of 5th digit - -Other Abnormalities Criptorquidia

Renal Malformations - -Umbilical hernia (poco frecuente) - +

Materials and Methods

Cytogenetic analysis. Cytogenetic analysis with a resolution of 350-500 bands from lymphocytes of samples from the two siblings, the older brother, the parents, and the maternal aunt and uncle were performed according to standard techniques. Chromosomes were analyzed with routine karyotype, GTG-banding, NOR banding, CGB and FISH. For the FISH analysis the single copy LSI TUPLE 1 and ARSA (VYSIS) probes were used as a control for regions 22q11.2 and 22q13.

Microarray analysis

Peripheral blood samples were collected and DNA was extracted using the Qiagen Maxi Kit according to manufacturers instructions (Qiagen Inc., Hidelberg, GE). The concentration of genomic DNA for all samples was measured using a spectrophotometer (NanoDrop ND-1000, NanoDrop Technologies). Genomic DNA integrity was evaluated with a 1% agarose gel electrophoresis. Samples were adjusted to a 50 g/l concentration for further microarray processing . Microarray analysis was performed using the Affymetrix Genome-Wide Human SNP Array 6.0 that includes a total of 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 copy number variation probes. Array processing and hybridization to was performed using the Genechip reagents kit and manufacturers instructions (Affymetrix, Santa Clara, CA). Briefly, 250 ng of genomic DNA was digested with Sty I and Nsp I (New England BioLabs) and then ligated to Sty and Nsp specific adaptors. A generic primer that recognizes the adaptor sequence was used to amplify the DNA fragments. DNA fragments are PCR amplified in a GeneAmp PCR System 9700 (Applied Biosystems). PCR products from both reactions were pooled and purified with magnetic beads (Agencourt Magnetic Beads, Beckman). Concentration was measured with a Multiskan Spectrum (Thermo,Inc) and only samples with a concentration over 3.5 g/l were further fragmented and labeled with biotin. Hybridization was done for 16 hrs at 50℃in the Affymetrix GeneChip Hybridization Oven 640. Microarrays were washed and stained with a streptavidin-phycoeritrin solution (Molecular Probes, Invitrogen) using the Genechip Fluidics Stations 450 (Affymetrix). The Genechip arrays were scanned using the Affymetrix 3000 7G scanner and GeneChip Operating Software version 1.4 was used to generate the .CEL intensity files. Quality control of all microarrays were evaluated with the Genotyping Console version 3.0.2.

Analysis of CNVs

Genotyping Console 3.0.2 was used to assess quality control of the microarray experiments. Results for Call rate and Contrast QC were the main parameters considered. Genotyping was performed using the Birdseed v2 algorithm. Allele copy number variation and segmentation was calculated with the Partek Genomics Suite software, version 6.4 (Copyright© 2008, Partek Inc., St. Louis, MO, USA). Copy number and allele differences were obtained by an unpaired analysis , comparing the test samples with a baseline generated from 75 healthy mexicans samples genotyped with the same microarray platform. Allele Intensity import for baseline and problem samples was done using the same parameters, so fragment length correction was performed by Partek algorithm to avoid bias. The Partek genomic segmentation algorithm detects segments in neighboring regions where there is statistically significant

(p value treshold <0,001) difference in average intensity. A minimum amount of 10 markers were considered for segmentation results.

Cytogenetic abnormalities detected in the family members.

All the metaphases studied in the proband and in her brother (proband 2), showed a partial trisomy in chromosome 9 from 9pter to 9q13. GTC karyotype result was 46,XX +der(9)t(9;22)(q13;q11)mat,-22. FISH analysis results were 46,XX,ish22q11.2 (D22S533,D22S942x2), 22q13.3 (ARSAx2) (ISCN,2005). The sample from the father and older brother showed a normal karyotype. The karyotype of the mother showed that she is a carrier of a balanced translocation (9;22)(q13;q11) (Figure 1). In the clinical interview, where the mother stated the there was a possibility of finding other carriers of the translocation in the family, a cytogenetic analysis was performed to samples from the sister and brother (maternal line aunt and uncle) and they are also carriers of the traslocation. A detailed interview to the members of the family was performed and it was inferred that the grandmother and great-grandfather were also obligatory carriers of the translocation.

Microarray results

CNV differences detected in the family.Considering Genotyping Console parameters , all the samples hybridized to the Genechip SNP 6.0 array complied with quality control parameters (average QC call rate for the samples was 96,78 and Contrast QC average value was 2,49). The segmentation analysis showed a total of 193 copy number variant segments (CNVs) for the mother, 247 CNVs for the father, 221 CNVs in proband 1 (daughter) and 222 CNVs in the proband 2 (son). Using microarray analysis, partial trisomy of chromosome 9 was detected from cytoband 9p24.3 to 9q12 in both probands consistent with the karyotype results. However, in proband 1 two deletions are detected in the trisomic region. The first deletion, a 192.5 Kb segment, is located in cytoband 9p11.2 (start position 43533090 to 43725623) and contains the FAM75A6 gene. The other deleted segment is a 25,45 Kbs region that

Differences detected between proband1 and proband 2.Along with the 39.4 Mb amplification in chromosome 9 starting at There were 11 CNV segments detected in the samples of the 2 affected siblings only (Table 1).

Table 1. CNVs detected in Proband 1 and Proband 2.Chromosome Band Length Type of

alterationGenes

2 2p12 2.16 Kb Deletion No genes 3 3p12.3 2.21 Kb Amplification No genes 4 4p16.1 19.8 Kb Deletion No genes

4 4p11 250 Kb Deletion No genes

5 5q13.2 1.02 Mb Deletion SERF1A,SERF1B,SMN1,SMN2,GTF2H2C,GTF2H2B,GTF2H2D.

10 10q22.1 1.06 Kb Deletion KIAA127411 11q11 66.8 Kb Amplification OR4S214 14q11.1 638.3 Kb Deletion OR11H12, POTEG17 17p13.3 5.75 Kb Deletion LOC28400917 17q21.31 26.9 Kb Deletion LRRC37A, ARL1722

22q11.1450.99 Kb Deletion POTEH, OR11H1

The 1.02 Mb deletion of 5q13.2 is one of the interesting aberrations detected in both affected children. (REVISAR GENES DE LA REGION)This telomeric region is part of a 500Kb inverted duplication, containing four genes (SERF1A,SERF1B,SMN1,SMN2) and repetitive elements, which make it prone to rearrangements and deletions. Smn1 and Smn2 genes code for the survival motor neuron protein (SMN), a molecule with

Many of these alterations are located in duplicated regions, prone to rearrangements and other are known polymorphic variants such as the olfactory receptor genes (OR4S2, OR11H12, OR11H1)

CNVs detected only in Proband 1 (severely affected child)A total of 51 CNVs were detected only in Proband 1, the patient with the severe mental and developmental delay (Table 2).